Recombinant extracellular chitinase from brevibacillus laterosporus for biological control and other industrial uses

ABSTRACT

The present invention discloses a recombinant modified extracellular chitinase having an amino acid sequence set forth in SEQ ID NO.4 or SEQ ID NO.5 prepared by substituting two tyrosine (Y) residues with histidine (H) in the native chitinase of  Brevibacillus laterosporus  LAK 1210. The modified chitinase represents an advancement as it has improved thermal stability, wider optimum pH range, high efficacy, improved solubility and low toxicity. The present invention also provides compositions and improved methods for producing and purifying the recombinant modified chitinase by chitin affinity chromatography and chitin adsorption affinity chromatography using shrimp shell and crab shell, for large scale production at low cost. The modified chitinase has wide range of applications including prevention, treatment or modulation of phytopathogenic infection in a plant or a plant part.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 16/122,612, filed Sep. 5, 2018, which claims priority from IndianPatent Application No. 201711031399, filed Sep. 5, 2017.

TECHNICAL FIELD

The present invention relates to the field of enzyme biotechnology, morespecifically a novel chitinase enzyme-based technology forbiotechnological applications in agriculture, environment andbiomedicine. The present invention pertains to a recombinantextracellular chitinase from Brevibacillus laterosporus LAK 1210 forbiological control and its other industrial uses.

BACKGROUND

Typically, plant diseases caused by phytopathogenic organisms includinginsects and fungi are a major constraint on forest and agriculturalproductivity. The use of conventional pesticides for plant protection isbeing undermined by problems related to insect resistance, pestresurgence and environmental concerns. Copping et al (2000) havereported that a variety of insect pests cannot be effectively controlledwith available pesticide regimens (Copping, L. G and Menn, J. J (2000)Biopesticides: a review of their action, applications and efficacy. PestManag. Sci 56(8):651-676). Chandler D, Bailey A S, Tatchell G M,Davidson G, Greaves J, Grant W P (2011) The development, regulation anduse of biopesticides for integrated pest management. Phil. Trans. R.Soc. B. Biol. Sciences 366(1573):1987-1998). Matyjaszczyk E (2015)Products containing microorganisms as a tool in integrated pestmanagement and the rules of their market placement in the EuropeanUnion. Pest Manag. Sci. 71:1201-1206).

In principle, biopesticides may effectively address most of thechallenges related to the use of pesticides and as a result theyreceived ample attention in current research programs on sustainablecrop protection. Development and commercial implementation of novelbiopesticides has been actively encouraged to realize the utility ofeffective new pest management solutions. A number of biopesticidescurrently available for the control of insect pests and phytopathogenicfungi have debatable effectiveness. Though Bacillus thuringiensis hasbeen widely used to combat destructive pests, the development ofresistance to Bt-insecticides is a clear threat. (Bruce E Tabashnik,Thierry Brévault and Yves Carrière (2013) Insect resistance to Bt crops:lessons from the first acres. Nature Biotechnology 31: 510-521).

The development of insect resistance to Bt necessitates an in-depthcontinuing search for new biocontrol agents having activity against awide variety of insect pests and improved insecticidal activity. Therehave been few effective microbial insecticides since Bacillusthuringiensis (Bt) and there is a quest for novel strains as alternativeto Bt control. As an alternative certain naturally-occurring agents havebeen isolated and developed as pesticides. These include naturalstrains, novel polypeptides and proteins. Consequently, there is a greatinterest and utility in finding natural strains and polypeptides withdeleterious effect on insect pests and phytopathogenic fungi.

Current and future regimes in integrated pest management would benefitfrom an intensified focus on enzyme applications for biological pestcontrol. An interesting application of chitinolytic enzymes is inbiological control of insect pests and phytopathogenic fungi.(Herrera-Estrella A and Chet I (1999) Chitinases for biological control.EXS: 87:171-84). The implication of role of chitinases in biocontrol hasbeen investigated and the approach to chitinolytic enzymes forbiocontrol of fungal and insect pathogens is based on the widespreadpresence of chitin as an integral part of the cell walls of fungi,insect cuticle and crustacean exoskeletons. Chitin present in thecuticle and midgut of insects, cell wall of phytopathogenic fungi willbe suitable targets for disruption and perturbation by chitinolyticenzymes. Hence, highly effective chitinases with a rationale ofdeveloping chitinase-based biocides that interfere with the chitinbiosynthesis in insects and phytopathogenic fungi perfectly fit a modernbiopesticide/biocontrol agent with desired efficacy and safety profile.Till date, very few bacterial chitinases with antifungal or insecticidalactivity have been identified and biochemically characterized. Theresearch reports are available for the synergistic action of chitinasesto potentiate the insecticidal activity of Bt toxins. The low efficiencyand high production and purification costs limited the development andcommercial use of chitinases as biocides.

Chitin is the main structural component of the fungal cell wall and theexoskeletons of invertebrates, such as insects and crustaceans. Chitinis an insoluble homopolymer of β-(1,4)-linked N-acetylglucosamine(GlcNAc), is the second most abundant polysaccharide in the biosphere,next to cellulose. Chitinases are glycosyl hydrolases that catalyze thehydrolytic cleavage of the β-1,4-glycoside bond and are found inincluding microbes, plants, insects, and mammals (Fukamizo T (2000)Chitinolytic enzymes: catalysis, substrate binding, and theirapplication. Curr Protein Pept Sci 1:105-124). Chitinases mediate thedegradation of chitin sources in the nature and also the digestion ofchitin present in the exoskeleton and peritrophic membrane (PM) in themidgut of insects to soluble chitooligosaccharides (Gooday G W (1999)Aggressive and defensive roles for chitinases. EXS: 87:157-69). YasuyukiArakane, Toki Taira, Takayuki Ohnuma and Tamo Fukamizo (2012)Chitin-Related Enzymes in Agro-Biosciences, Current Drug Drug Targets13(4): 442-470).

In recent years, significant research has been directed toward the useof chitinolytic enzymes with potential applications in fields as diverseas plant protection, bioremediation, effluent water treatment and drugdelivery. (Chavan S B, Deshpande M V (2013) Chitinolytic enzymes: anappraisal as a product of commercial potential. Biotechnol Prog.29(4):833-46).

Microorganisms, in particular bacteria are the major source of mostindustrially important chitinases (Qiang Yan and Stephen S Fong (2015)Bacterial chitinase: nature and perspectives for sustainablebioproduction. Bioproc. and Bioeng. 2 (31) 1-9). Some of the best knownchitinolytic bacteria include Serratia, Bacillus, Aeromonas, Vibrio, andStreptomyces. Chitinases are also reported from other bacterial specieslike Enterobacter, Pseudomonas, Alcaligenes and Paenibacillus. With thegrowing need for green alternatives to industrial processes, chitinaseshave paved a way for their efficient utilization with new possibleapplications in biorefinery, single-cell protein production, as a foodquality enhancer and for the control of malaria propagation (Bae KeunPark and Moon-Moo Kim (2010) Applications of Chitin and Its Derivativesin Biological Medicine. Int. J. Mol. Sci. 11 5152-516). Their versatileapplications prompted the discovery of new strains that are capable ofproducing chitinolytic enzymes with novel properties. There is acontinuous search for new and novel chitinolytic enzymes withcharacteristics more suitable for biological control and otherindustrial applications.

A concerted understanding of structure and function of bacterialchitinases from new strains and organisms will be necessary foradvancing chitinase research toward biological control and other novelbiotechnological applications. In the current scenario, isolated,thermostable enzymes are preferred over microorganisms for industrialapplications, since, unlike many microbes, enzymes remain effective in awide range of pH and temperature range. Secretion of recombinant enzymesto extracellular milieu is important for the successful use of cheap,efficient and thermostable biomass hydrolyzing enzymes for biorefineryto convert plant biomass to biofuels. There is a search forextracellular chitinolytic enzymes that could enhance bioremediation ofrecalcitrant compounds and effluent water. Therefore, secretion ofexpressed recombinant enzyme into the culture medium can be a solutionfor the large-scale production of recombinant E. coli for applicationsin such industrial bioprocesses Furthermore, thermostable enzymes areresistant to organic solvents, detergents, denaturing agents and bettersuited for harsh industrial processing conditions in terms of thermalactivity and stability. Some extracellular enzymes isolated frommesophiles might be active at considerably higher temperatures thantheir host's environments.

Brevibacillus laterosporus is an emerging entomopathogen and itsinsecticidal activity has been reported against Lepidoptera, Coleoptera,Diptera and nematodes (Oliveira E J D, L Rabinovitch L, Monnerat R G,Passos L K J and Zahner V (2004) Molecular Characterization ofBrevibacillus laterosporus and its Potential Use in Biological Control.Appl. Environ. Microbiol. 70 (11): 6657-64).

Though Brevibacillus laterosporus is reported to have a wide spectrum ofbiological activity compared to the most popular entomopathogenicbacteria, Bacillus thuringiensis and Bacillus sphaericus, its biologicalcontrol potential has not been fully explored since the attempts toisolate this organism from different ecological niches was notsuccessful since the distribution of strains of Brevibacilluslaterosporus is limited compared to the strains of Bacillusthuringiensis and Bacillus sphaericus.

There are very few well documented reports available on the potentialuse of the entomopathogenic bacterium, Brevibacillus laterosporus as aneffective biopesticide/biocontrol agent. (Luca Ruiu (2013) Brevibacilluslaterosporus, a pathogen of Invertebrates and a Broad-SpectrumAntimicrobial Species. Insects 4: 476-492). However, thus far, there areno reports available from the patent and non-patent literature on theuses and commercial exploitation of chitinases from Brevibacilluslaterosporus. A small number of research articles have been publishedabout the enzymatic profile and effects of toxicity and antagonisticactivity from Brevibacillus laterosporus strains (Huang X, Tian B, NiuQ, Yang J, Zhang L, Zhang K (2005) An extracellular protease fromBrevibacillus laterosporus G4 without parasporal crystals can serve as apathogenic factor in infection of nematodes. Res. Microbiol. 156(5-6):719-727).

Shanmugiah et al (2008) have reported the identification and optimizedculture conditions for the production of chitinase from Bacilluslaterosporus MML2270 but chitinolytic activity and insecticidal activityhas not been reported for the said strain (Shanmugaiah V, Mathivanan N,Balasubramanian N and Manoharan P T (2008) Optimization of culturalconditions for production of chitinase by Bacillus laterosporus MML2270isolated from rice rhizosphere soil. African Journal of BiotechnologyVol. 7 (15): 2562-2568). Sakia et al reported a strain of Brevibacilluslaterosporus with antibacterial and antifungal compounds and notchitinolytic activity (Saikia R, Gogoi D K, Mazumder S, Yadav A, Sarma RK, Bora T C, Gogoi B K (2011) Brevibacillus laterosporus strain BPM3, apotential biocontrol agent isolated from a natural hot water spring ofAssam, India. Microbiol. Res., 166: 216-225).

Liu et al (2005) have reported thermotolerant chitinases (Chi A and ChiC) from Brevibacillus laterosporus M64, with a pH optimum of 7.0 and 6.0and thermal stability up to 55° C. (Pulin Liu, Deyong Cheng and LihongMiao (2015) Characterization of Thermotolerant Chitinases Encoded by aBrevibacillus laterosporus Strain Isolated from a Suburban Wetland.Genes 6: 1268-1282). U.S. Pat. No. 5,045,314A discloses the use ofinsecticidal strain of Bacillus laterosporus against nematodeova/larvae. Ignazio Floris et al (2008) reported an invention whichrelates to a new bacterial strain used for biological control ofinsects, especially Dipters (Ignazio Floris, Luca Ruiu, Alberto Satta,Gavino Delrio, Salvatore Rubino, Bianca Paglietti, David John Ellar,Roberto A. Pantaleoni (2008) Brevibacillus laterosporus straincompositions containing the same and method for the biological controlof dipters (WO2008031887 A2) and published their results on lethaleffects of Brevibacillus laterosporus on Musca domestica. WO200831887 A2discloses the use of an insecticidal strain of Brevibacilluslateropsorus effective against dipterans (mosquitoes and mosquitolarvae) and this strain also has been reported to produce onlyinsecticidal proteins, with no chitinase activity. Traves Robert Glareet al reported an invention (WO 2014045131) which discloses theinsecticidal activity of the spore toxins of the three new strains ofBrevibacillus laterosporus against plant pests, particularly,Lepidoptera and Diptera. (Travis Robert Glare, John Graham Hampton,Murray Paul Cox, Damian Alexander Bienkowski (2014) Novel strains ofBrevibacillus laterosporus as biocontrol agents against plant pests,particularly lepidoptera and diptera (WO 2014045131).

However, usage of chitinases for efficacious pest management andindustrial applications have not been successful due to the followingfactors:

-   -   low expression level of chitinases in native hosts    -   additional costs in large-scale production and purification of        the recombinant enzyme    -   bioconversion activity being restricted to a narrow pH and        temperature range    -   lack of thermostability    -   toxicity to non-target species    -   low efficacy and lack of wide spectrum insecticidal and        fungicidal activity

WO2013050867 A2 provides a biologically pure culture of a new strain ofBrevibacillus laterosporus designated as Lak 1210 (MTCC 5487) having anaccession no 5487, as a dual producer of insecticidal proteins andinducible chitinolytic enzymes with a potential utility in agricultureand forest pest management, plant disease control and mosquito controlprograms. The strain Brevibacillus laterosporus Lak 1210 (MTCC 5487) isa novel, chitinolytic, insecticidal strain of Brevibacillus laterosporusand its potential insecticidal activity and antifungal activity has notbeen documented, till it was reported by Prasanna et al. (Prasanna, L,Eijsink, V. G. H, Meadow, R, Gaseidnes, S (2013). A novel strain ofBrevibacillus laterosporus produces chitinases that contribute to itsbiocontrol potential. Appl. Microbiol. Biotechnol. 97: 1601-1611) and(Lakshmi Prasanna, G (2012). A chitinase from Brevibacilluslaterosporus, its production and use thereof. WO 2013050867 A2). WO2013050867 A2 discloses that the multi-chitinolytic complex from theBrevibacillus laterosporus Lak 1210 has shown excellent chitindegradation ability, insecticidal activity against lepidopteran insects(diamond backmoth) as well as antifungal activity against severalphytopathogenic fungi. The strains of Brevibacillus laterosporus arerarely distributed and more specifically hyperchitinolytic strain ofBrevibacillus laterosporus is a rare find, the subject for the presentinvention is to isolate one of the chitinases from the multichitinolyticcomplex of the newly discovered strain, Brevibacillus laterosporus Lak1210 and clone it in E. coli to obtain a recombinant, extracellularchitinase for biocontrol and other industrial applications.

In the present invention, the inventors have identified the issues inprior art and have contemplated a unique enzyme engineering approachwherein a modified recombinant chitinase enzyme from Brevibacilluslaterosporus Lak 1210 has been invented. The present invention overcomesthe technical problems involved in large scale production andcost-effective purification of chitinases for industrial use, existingin the prior art. Apart from several other advantages, the recombinant,modified enzyme is thermoactive (55-60° C.) with high thermo-stability(66.7° C.), can operate at a wide pH range (pH 3.0-11.0) with analkaline pH optimum of 9.0. Because of the combined exo- andendochitinase activity and its alkaline pH optimum, the recombinantchitinase is highly efficacious in insecticidal knockdown both as acontact biopesticide and ingestion biopesticide. as compared to theother chitinolytic enzymes for biocontrol, existing in the prior art. Itis also desirable to expand the insecticidal activity of Bacillus-basedpesticides with addition of recombinant, modified chitinase for animproved pesticidal activity, where a wider range of pests are impacted.

The present invention overcomes the problems of the prior art to solve along-standing problem of lack of large scale production and large scale,cost-effective purification methods to produce a highly efficient,industrial chitinase for commercial applications in agriculture,medicine and environment.

SUMMARY

The present invention extends the current state of the art byengineering a chitinase from Brevibacillus laterosporus Lak 1210 in E.coli and the use of recombinant chitinase for biocontrol and proposedindustrial applications in agriculture, medicine and environment.

Since, Bt-based insecticide formulations have to be ingested and thetiming is critical to ensure that the bacterial toxins remain stable inthe environment until they are ingested by the target insect stage,there is a continuous search for new strains with novel mode of action.The present invention provides a recombinant, modified chitinase whichis novel properties which could be an effective protein biopesticide forcontrolling wide range of insect pests and phytopathogenic fungi andfurthermore, with possible industrial applications in agriculture,medicine and environment.

A full length chitinase gene (2583 bp) with a signal peptide of 42 aa(SEQUENCE ID NO: 1) from Brevibacillus laterosporus Lak 1210 (depositedunder MTCC Accession No. 5487) was mutated at positions 661 and 2158 bysite directed mutagenesis. The mutated nucleotide sequence (SEQUENCE IDNO: 3) was cloned in E. coli BL 21 (DE 3) for secreted expression of therecombinant, modified chitinase. The recombinant, modified chitinasecarries an amino acid substitution at positions 221 and 720, wherein,the tyrosine was replaced by histidine (example 1 and 2).

The recombinant, modified chitinase enzyme, designated as BRLA_Chi90 issecreted into the extracellular medium making it suitable for largescale production required for several industrial applications ofchitinase, which will increase the commercial feasibility of the enzyme(example 3).

Based on the examples, the supporting figures and drawings of thepresent invention claims a new, non-obvious method of chitinaseenzyme-based technology for biological control and industrial uses, theimprovements in the production and purification processes and noveltyand advantages of the recombinant, modified chitinase comprising

Novel purification protocol by chitin adsorption affinity chromatographyusing powdered crustacean shells making the large scale purificationstrategy simple and cost-effective (example 3).

The recombinant, modified chitinase enzyme is thermoactive (optimum pH55-60° C.) thermostable (66.7° C.) (FIG. 11 from the description ofdrawings) active over a broad range of pH (3.0-11.0) with a pH optimumof 9.0, suitable for several commercial biotechnology applications likebioremediation, biorefinery, where the industrial operations use highlystable, enzyme that can withstand harsh industrial environment, provingthat its truly an industrial chitinase (example 8, 9 and 10).

The recombinant, modified enzyme exhibits dual enzyme activity (bothexo- and endochitinase activity) is a very important feature which canmake it a more efficacious biocontrol agent with enhanced biocontrolpotential against insect pests and phytopathogenic fungi (example 10).

Substitution of tyrosine with histidine results in improved solubilitywhich improves the amphipathic nature and physiological action of thechitinase, while acting on the chitin in the cuticle and pore formationin the chitin-lined midgut of the larvae to enhance the membranepermeability and allow the toxins to permeate through (example 6).

Contact bioassays followed by the SEM and histopathological studies havedemonstrated insecticidal knockdown which results in mortality of thetarget insects in 48 h. The recombinant, modified chitinase fromBrevibacillus laterosporus Lak 1210 can be developed into a novel,contact biopesticide due to its mode of action on the insect cuticle bythe novel enzyme-based technology (example 11 and 12).

Contact insect bioassays have also demonstrated that the modification ofprotein helps the enzyme better to penetrate the insect cuticle hasresulted in increased efficacy for degrading the cuticle during molting,resulting in the death of larvae and also delay in adult emergence(Example 17).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: PCR amplification of BRLA_Chi 90 gene from Brevibacilluslaterosporus Lak 1210 Lane 1—DNA marker—1 Kb ladder and Lane 2—2583 bpPCR product of Blchi gene.

FIG. 2A: SDS-PAGE analysis of recombinant, modified BRLA_Chi 90 fromBrevibacillus laterosporus Lak 1210: M13 Protein marker, 1. lysate, 2.periplasmic fraction 3. culture supernatant 4. soluble fraction, 5.insoluble fraction, 6 and 7. chitinase fractions.

FIG. 2B. Chitinase zymography with 4-Methylumbelliferyl substrates usingrecombinant, modified BRLA_Chi 90 from Brevibacillus laterosporus Lak1210: 1. N-acetylglucosaminide 2. 4 MU—chitobioside 3. 4MU—chitotrioside). The numbers on the left dictate the molecular masses(in kilodaltons) of the protein standards. The positions of the purifiedchitinase fraction is indicated by arrows.

FIG. 3: Secretion profile of recombinant, modified chitinase BRLA_Chi 90from Brevibacillus laterosporus Lak 1210 expressed in the different cellcompartments—samples of the extracellular medium (▪) periplasm (●) inthe cytoplasm (▴) from 24-h time points of E. coli BL 21 (DE3). Theβ-galactosidase activity detected in the culture medium (♦) was used asan indicator of cell lysis. The culture was grown at 37° C. until OD₆₀₀reached 0.8, induced with 0.1 mM IPTG and incubated for another 24 h.All experiments were evaluated under identical conditions and performedin triplicate.

FIG. 4: Multiple sequence alignment comparison of the 90 kDarecombinant, modified chitinase, BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210 with the conserved domains of chitinases fromentomopathogens.

FIG. 5: Multiple sequence alignment comparison of the 90 kDarecombinant, modified chitinase BRLA_Chi 90 of from Brevibacillus.laterosporus Lak 1210 with the conserved domains of chitinases fromother bacterial chitinases.

FIG. 6: MALDI-TOF/MS spectrum of purified recombinant, modified BRLA_Chi90 from Brevibacillus laterosporus Lak 1210. The molecular mass of theenzyme was 89680 Da.

FIG. 7: Optimum temperature profile of recombinant, modified chitinase,BRLA_Chi 90 from Brevibacillus laterosporus Lak 1210. Assayconditions-Assay buffer-sodium acetate (pH 5.0), reaction volume (100μl) substrate concentration (10 μl), enzyme concentration (0.1 μg).

FIG. 8: Optimum pH profile of recombinant chitinase, BRLA_Chi 90 fromBrevibacillus laterosporus Lak 1210.

Assay conditions—buffer systems used were citrate buffer (50 mM, pH3.0-4.0), acetate (50 mM, pH 4.0-5.0), phosphate (50 mM, pH 6.0-8.0),Tris HCl (50 mM 8.0-9.0) and carbonate-bicarbonate (50 mM, pH 9.0-11.0),reaction volume (100 μl) substrate concentration (10 μM), enzymeconcentration (0.1 μg).

FIG. 9: CD spectrum of recombinant, modified BRLA_Chi 90 fromBrevibacillus laterosporus Lak 1210 showing secondary structure with20.47% alpha helix, 27.09% beta sheets, 11.57% turns and 40.93% coils.

FIG. 10: CD spectrum of thermal unfolding of recombinant, modifiedchitinase, BRLA_Chi 90 from Brevibacillus laterosporus Lak 1210.

FIG. 11: Mass spectroscopic analysis-using electrospray ionization massspectrometry (ESI-MS) of chitooligosaccharides standards showing theirmasses GlcNAc (244.11), GlcNAc2 (447.23) GlcNAc₃ (650.30), GlcNAc4 (853.35), GlcNAc₅ (1056.38) and GlcNAc6 (1259.4).

FIG. 12: ESI-MS profile for time course analysis of chitin hydrolysisusing recombinant, modified BRLA_Chi 90 from Brevibacillus laterosporusLak 1210 (20 min).

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Chitooligomers obtained from colloidal chitin after digestion (20 min)depicted two peaks corresponding to GlcNAc₃ (m/z 650.83) a sodiumadducts of three NAG molecules (chitotriose) and GlcNAc₂ (m/z 447.2), asodium adducts of two NAG molecules (chitobiose).

FIG. 13: ESI-MS profile for time course analysis of chitin hydrolysisusing recombinant, modified BRLA_Chi 90 from Brevibacillus laterosporusLak 1210 (1 h).

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Intensity of GlcNAc₂ increased with time. Apparently, GlCNAc₃ was alsocleaved to form GlcNAc₂.

FIG. 14: ESI-MS profile for time course analysis of chitin hydrolysisusing recombinant, modified BRLA_Chi 90 from Brevibacillus laterosporusLak 1210 (3 h).

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

After 3 h, the product is predominantly GlCNAc₂. No higher oligomersdetected.

FIG. 15: ESI-MS profile for time course analysis of chitin hydrolysis (6h) using recombinant, modified BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210.

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Chitooligomers obtained from colloidal chitin after digestion (6 h)GlcNAc₃ (m/z 650.83) sodium adducts of three NAG molecules (chitotriose)and GlcNAc₂ (m/z 447.25), a sodium adducts of two NAG molecules(chitobiose).

FIG. 16: ESI-MS profile for time course analysis of chitin hydrolysis(12 h) using recombinant, modified BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210.

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Chitooligomers obtained from colloidal chitin after digestion (12 h),only GlcNAc₂ (m/z 447.25), a sodium adduct of two NAG molecules(chitobiose) was detected

FIG. 17: ESI-MS profile for time course analysis of chitin hydrolysis(24 h) using recombinant, modified BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210.

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Chitooligomers obtained from colloidal chitin after digestion (24 h) andN-acetylglucosamine (m/z 226.86) was also a major product along with theGlcNAc₂ (m/z 447.25), a sodium adducts of two NAG molecules(chitobiose).

FIG. 18: ESI-MS profile for time course analysis of chitin hydrolysis(48 h) using recombinant, modified BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210.

Reaction conditions: Substrate (colloidal chitin) 10 mg/ml, purifiedchitinase 100 μg, reaction volume-100 μl, reaction buffer-50 mM sodiumacetate (pH 5.0).

Chitooligomers obtained from colloidal chitin after digestion (48 h) andN-acetylglucosamine (m/z 226.86) was predominantly detected along withthe chitobiose (m/z 447.25) indicating that the chitobiose was cleavedto give N-acetylglucosamine as the final product of chitin hydrolysis.

FIG. 19A: Scanning Electron Microscopy (SEM) study of hydrolytic effectsof the recombinant, modified chitinase BRLA_Chi 90, on an untreatedcuticle of Spodoptera litura (topical bioassays);

FIG. 19B: SEM study of hydrolytic effects of the recombinant, modifiedchitinase BRLA Chi 90, on a treated cuticle (12h) of Spodoptera litura(topical bioassays);

FIG. 19C: SEM study of hydrolytic effects of the recombinant, modifiedchitinase BRLA_Chi 90, on a treated cuticle (24h) of Spodoptera litura(topical bioassays).

FIG. 20A: Histopathological study showing ultrastructural changes in theuntreated midgut (control) of Spodoptera litura following dropletfeeding insect bioassays in which the larvae were orally fed withrecombinant, modified chitinase, BRLA_Chi 90 from Brevibacilluslaterosporus Lak 1210;

FIG. 20B: Histopathological study showing ultrastructural changes in thetreated midgut (48 h) of Spodoptera litura following droplet feedinginsect bioassays in which the larvae were orally fed with recombinant,modified chitinase, BRLA_Chi 90 from Brevibacillus laterosporus Lak1210.

FIG. 21: Antifungal activity of recombinant, modified chitinase,BRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 against Fusariumoxysporum (hyphal extension inhibition assay).

FIG. 22A: Scanning Electron Microscopy (SEM) study of hyphal morphology(untreated hyphae) of Fusarium oxysporum treated with recombinant,modified chitinase, BRLA Chi 90 from Brevibacillus laterosporus Lak1210;

FIG. 22B: SEM study of hyphal morphology (treated hyphae, 6h) ofFusarium oxysporum treated with recombinant, modified chitinase,BRLA_Chi 90 from Brevibacillus laterosporus Lak 1210.

FIGS. 23A-23D: Localization of FITC-BRLA Chi 90 from Brevibacilluslaterosporus Lak 1210 in the hyphae of Fusarium oxysporum. Hyphae wereidentified based on the differential interference contrast (DIC) image.Subsequently, the localization of the GFP signal was scored in at least100 hyphae. FIG. 23A: Localization of FITC-labelled recombinantchitinase BRLA Chi 90 at the septa. FIG. 23B: Localization ofFITC-labelled chitinase in the cortex of mature hypha FIG. 23C:Localization of FITC-labelled recombinant chitinase on the cell wallFIG. 23D: Localization of FITC-labelled recombinant chitinase at thehyphal tip.

FIGS. 24A and 24B: Fluorescence microscopy study to evaluatelocalization of FITC-BRLA Chi 90 from Brevibacillus laterosporus Lak1210, in the hyphae of Fusarium oxysporum. FIG. 24A: Swelling of hyphaltip. FIG. 24B: Complete destruction of the hypha.

FIGS. 25A and 25B: Bright field photomicrographs of histology of heartin rats (40×, H & Estain) FIG. 25A: Control treated with sterile PBS.FIG. 25B: Test treated with 20 mg/kg of purified chitinase fromBrevibacillus laterosporus Lak 1210.

FIGS. 26A and 26B: Bright field photomicrographs of histology of kidneyin rats (40×, H & Estain) FIG. 26A: Control treated with sterile PBS.FIG. 26B: Test treated with 20 mg/kg of purified recombinant, modifiedchitinase BRLA Chi 90 from Brevibacillus laterosporus Lak 1210.

FIGS. 27 A and 27B: Bright field photomicrographs of histology of liverin rats (40×, H & E stain) FIG. 27 A: Control treated with sterile PBS,FIG. 27B: Test treated with 20 mg/kg of purified recombinant chitinaseBRLA Chi 90 from Brevibacillus laterosporus Lak 1210.

FIGS. 28A and 28B: Bright field photomicrographs of histology of lungsin rats (40×, H & E stain) FIG. 28A: Control treated with sterile PBS,FIG. 28B: Test treated with 20 mg/kg of purified recombinant chitinaseBRLA Chi 90 from Brevibacillus laterosporus Lak 1210.

FIGS. 29A and 29B: Bright field photomicrographs of histology of spleenin rats (40×, H & Estain) FIG. 29A: Control treated with sterile PB S,FIG. 29B: Test treated with 20 mg/kg of purified recombinant chitinaseBRLA Chi 90 from Brevibacillus laterosporus Lak 1210.

FIG. 30: Protein structure-function prediction of the substitution siteof the modified recombinant chitinase enzyme from Brevibacilluslaterosporus Lak 1210.

DETAILED DESCRIPTION

Development of bioinsecticides for use against insects has generallybeen limited to Bacillus thuringiensis and majority of the patents ininsect control are based on cry toxins of Bacillus thuringiensis. Theknowledge base on enzyme-based biological control is very little and itneeds increased scientific space in entomological and biotechnologicalresearch. The scope of the invention is to develop a novel chitinaseenzyme for controlling key insect pests and fungal diseases ofimportance agriculturally important crops, fruit and vegetable crops,natural forests and forest plantations and other industrialapplications. Strong contact toxicity through topical application of thenovel chitinase from the strain of Brevibacillus laterosporus Lak 1210has proved that it is toxic to the insect upon direct contact and mayresult in quick kill.

Nucleic acids described herein as “isolated” are nucleic acids that havebeen obtained from their natural source or separated from the genomicDNA using biological and/or and chemical methods. Nucleic acidsdescribed herein as “recombinant” are nucleic acids or fragments orvariants thereof which have been generated by molecular techniques suchas PCR, including conventional PCR, overlap extension PCR-mediatedsite-directed mutagenesis that alter or extend the gene coding sequenceby virtue of substitution of one or more nucleotides in theoligonucleotides utilized in the PCR amplification and/or cloning intoan expression vector using restriction enzymes. As described herein,“active form” means chemically active and capable of hydrolysing and orsolubilizing chitinous substrates and “biologically active” meanscapable of controlling insects and inhibiting phytopathogenic fungi.

In an embodiment of the present invention, an isolated nucleic acidsequence set forth in SEQUENCE ID NO:1 encoding chitinase having theamino acid sequence set forth in SEQUENCE ID NO:2 from Brevibacilluslaterosporus Lak 1210 deposited under MTCC Accession No. 5487 isdisclosed. The SEQUENCE ID NO:2 from position 1 to 42 is a secretorysignal peptide encoded by a sequence set forth in SEQUENCE ID NO:6. Thesignal peptide sequence has the amino acid sequence as set forth inSEQUENCE ID NO:40: MRKMYQHIPTAHSVRKFNFLLLAFVLFASIFPAILPASVSAS. Thesignal peptide sequence derived from the chitinase gene of the strain ofBrevibacillus laterosporus Lak 1210 has been found to function as astrong secretion signal when fused to gene sequences of the expressionvector pET 21b to construct a recombinant expression plasmid,pET/BLRA_Chi90 and expressed in the host cells. The signal peptide, whenfused to gene sequences and expressed as a polypeptide, localize therecombinant protein as periplasmic, cytoplasmic fractions and alsosecreted from the cell and recovered from the extracellular medium. Thenative secretory signal sequence of BLRA_Chi 90 gene of SEQUENCE ID NO:6 can be linked to foreign DNA for secreted expression of therecombinant protein from other organisms. The nucleotide sequences forthe native chitinase were deposited with the GenBank with the accessionnumber MF397932.

The present invention also includes a recombinant nucleic acid sequenceas set forth in SEQUENCE ID NO:3 from Brevibacillus laterosporus Lak1210 encoding recombinant chitinase having an amino acid sequence setforth in SEQUENCE ID:.4 or SEQUENCE ID:5. The nucleotide sequences forthe recombinant (mutated) chitinase sequence were deposited with theGenBank with the accession numbers MF 397933. Particular embodiments ofthe invention further provide isolated biocidal (e.g., insecticidalactivity and antifungal activity) polypeptides encoded by either a fulllength native or recombinant nucleic acid of the embodiments. Inparticular examples, biocidal proteins of the embodiments includefragments of full-length proteins and polypeptides that are producedfrom mutagenized nucleotide sequence designed to introduce one or morenucleotide residues that is not present in the corresponding nativesequence. The embodiments further provide mutant nucleotide sequencewhich confer improved or altered properties on the polypeptides of theembodiments to accomplish the object of mutation, for example, anenhanced insecticidal activity, antifungal activity and chitinhydrolyzing activity.

The recombinant nucleic acid with a nucleotide sequence (SEQUENCE IDNO:3) further encodes truncated polypeptide sequences having SEQUENCE IDNO:7 to SEQUENCE ID NO:39 as shown in Table 1 below:

TABLE 1 Listing of unique peptides from peptide massfingerprinting of recombinant, modified BLRA_Chi 90(SEQUENCE ID NO: 7-SEQUENCE ID NO: 39) SEQUENCE ID PROTEIN SEQUENCESEQ ID NO: 7 IEVTGFQLGDQNYPINPTLK SEQ ID NO: 8 NYDSTLVAPWLWNAEKSEQ ID NO: 9 NLTHINYAFAHVDSNNR SEQ ID NO: 10 VFLSTEDEQSIGAKSEQ ID NO: 11 AKDNQGLESEASQPLK SEQ ID NO: 12 DNQGLESEASQPLKSEQ ID NO: 13 GFQNVVGGTDGLWGK SEQ ID NO: 14 DENGKEEGAGSNPMWHAKSEQ ID NO: 15 NDGKGEYYMGSTLTK SEQ ID NO: 16 DHGIVNPVLTGTYK SEQ ID NO: 17GHFNLLTQWK SEQ ID NO: 18 DENGKEEGAGSNPMWHAK SEQ ID NO: 19 EEGAGSNPMWHAKSEQ ID NO: 20 YYMLTIASPSSAYLLR SEQ ID NO: 21 LDQASAEDEK SEQ ID NO: 22GEYYMGSTLTK SEQ ID NO: 23 NDGKGEYYMGSTLTK SEQ ID NO: 24 IISAGHTGPNVGGLKSEQ ID NO: 25 GEYYMGSTLTK SEQ ID NO: 26 GLMEGYNALLK SEQ ID NO: 27EEGAGSNPMWHAK SEQ ID NO: 28 GMESFQALK SEQ ID NO: 29 TIYTSGQQASYKSEQ ID NO: 30 GLMEGYNALLK SEQ ID NO: 31 YLVTDIPWK SEQ ID NO: 32IIGYFTSWR SEQ ID NO: 33 GMESFQALK SEQ ID NO: 34 INIGVPYYTR SEQ ID NO: 35VTTDTDTLPPEPATPCRPAGLYDSGV SEQ ID NO: 36 VAVTIPTWK SEQ ID NO: 37 GHEWTAKSEQ ID NO: 38 KYAVTDK SEQ ID NO: 39 VKLDQASAEDEK

In an aspect, SEQ ID NO:3 is a mutated sequence set forth in an isolatednucleic acid sequence set forth in SEQ ID NO:1. CATATG is therestriction site for the restriction enzyme Nde 1, the native chitinasesequence can be cut by the restriction enzyme Nde 1 at two places. Tofacilitate the cloning of complete ORF and ensure the translation of theORF into a functional protein, single point mutations have beenincorporated by site directed mutagenesis at the positions 661 and 2158to replace the nucleotide ‘T’ with the nucleotide ‘C’. The CATATG in theORF of the native sequence has been changed to CACATG. The recombinant(mutated) sequence ORF has the sequence CACATG. Though the base ‘T’ isreplaced with ‘C’ since both the codons, CAT and CAC code for the sameamino acid, Histidine, and hence there is no change in the functionalityof the protein.

The site directed mutagenesis resulted in the substitution of a polar,hydrophilic histidine (H) residue for a hydrophobic aromatic amino acidtyrosine (Y) at positions 221 and 720 in the SEQUENCE NO. 4 and SEQUENCENO. 5. The amino acid substitution may increase the interaction with thenegatively charges insect cuticle. The increase in positive charge iscrucial for binding negatively charged insect cuticle and subsequentlymove into the aqueous haemolyph and continue to induce cell lysis,resulting in the death of the target insect.

Histidine is interesting in that it is an ideal residue for proteinfunctional centers, most common amino acid in protein active or bindingsite, with a pKa near that of physiological pH. The variation in peptidesequence should be possible without losing its biological activity.Substitution of tyrosine with histidine did not affect the amphipathicnature and physiological action of the protein while acting on thechitin the cuticle and pore formation in the chitin-lined midgut regionto enhance the membrane permeability and allow the toxins to permeatethrough.

Contact insect bioassays have demonstrated that the modification ofprotein to better penetrate the insect cuticle has resulted in increasedefficacy for degrading the cuticle during molting, resulting in thedeath of larvae and also delay in adult emergence.

Soluble proteins tend to have polar or hydrophilic residues on theirsurfaces. The aminoacid substitution at position 221 replacing tyrosinewith histidine resulted in improved solubility of the protein, anotherimportant desirable characteristic feature that enhances the efficacy ofa biocontrol agent/biopesticide and is much suitable for variousindustrial applications of chitinase.

In an aspect of the present invention, a DNA construct including theisolated nucleic acid sequence set forth in SEQUENCE ID NO:1 encodingnative chitinase having the amino acid sequence set forth in SEQUENCE IDNO:2 from Brevibacillus laterosporus Lak 1210 deposited under MTCCAccession No. 5487 is provided. In another aspect of the presentinvention, a DNA construct including the recombinant nucleic acidsequence as set forth in SEQUENCE ID NO:3 from Brevibacilluslaterosporus Lak 1210 encoding recombinant, modified chitinase havingamino acid sequence set forth in SEQUENCE ID NO:4 or SEQUENCE ID NO 5 isprovided.

In a further aspect, an expression vector including the DNA constructhaving the recombinant nucleic acid sequence as set forth in SEQUENCE IDNO:3 from Brevibacillus laterosporus Lak 1210 encoding recombinantchitinase having amino acid sequence set forth in SEQUENCE ID NO:4 orSEQUENCE ID NO 5 is provided. The expression vector includes therecombinant nucleic acid sequence set forth in SEQUENCE ID NO:3 fromBrevibacillus laterosporus Lak 1210 encoding chitinase having the aminoacid sequence set forth in SEQUENCE ID NO:4 or SEQUENCE ID NO:5,operably linked with T7 promoter and a native regulator sequence setforth in SEQUENCE ID NO: 6. The expression vector carrying the isolatednucleic acid sequence (SEQUENCE ID No 1) or recombinant nucleic acidsequence (SEQUENCE ID NO 3) designated as pET/BRLA_Chi90.

Truncated polypeptides of SEQUENCE ID No: 7 to SEQUENCE ID No: 39encoded by the polynucleotide fragments of the embodiments arecharacterized by insecticidal and antifungal activity that is eitherequivalent to, or improved, relative to the activity of thecorresponding full-length polypeptide of SEQUENCE ID No: 2, SEQUENCE IDNo: 4 and SEQUENCE ID No 5 encoded by the nucleic acid sequence ofSEQUENCE ID No: 1 and 3 from which the fragment is derived.

Generally, purification of chitinolytic enzymes is a multi-step processexploiting a range of biophysical and biochemical characteristics suchas its relative concentration in the source, solubility, charge, size(molecular weight), hydrophobicity/hydrophilicity of the target protein.In general, purification strategy is focused on high yield and recovery,purity of the enzyme, reproducibility of the method, economical use ofthe chemicals and reagents and shorter time for complete purification.

In an aspect, the present invention provides a single step purificationmethod by chitin affinity chromatography. The soluble, activerecombinant, modified chitinase BRLA_Chi 90 from the extracellularmilieu of E. coli can be purified by a single step, large scale,cost-effective chitin adsorption affinity purification method usingcolloidal chitin or fishery waste from sea food processing industry likecrustacean shells (crab shells, shrimp shells and krill shells). Therecombinant, modified chitinase of the present invention can bebiologically pure and can have a molecular weight of 89.68 kDadetermined by SDS-PAGE and MALDI-TOF (FIG. 2A). The aminoacid sequenceof the recombinant chitinase expressed as a mature protein (withoutsignal peptide) was listed as SEQUENCE ID NO: 5.

Based on the cleavage pattern, chitinases have been classified into twobroad categories i.e. endochitinases and exochitinases. Endochitinases(EC 3.2.1.14) randomly cleave β-1,4-glycosidic bonds of chitin polymerto produce oligomers of different length which can be converted intomixtures of dimers (diacetylchitobiose) and monomeric units(N-acetylglocosamine). Exochitinases are divided into two subcategoriesbased on the type of the product released:exo-N,N′-diacetylchitobiohydrolases, also designated as chitobiosidases(EC 3.2.1.29) which catalyze the progressive release of the dimer,diacetylchitobiose (GlcNAc2) from the non-reducing end of the chitin andβ-1-4 N-acetyl-D-glucosaminidase (EC 3.2.1.30) which cleave theoligomeric products of endochitinases and chitobiosidases, typicallychitobioses generating N-acetylglucosamine. They hydrolyze GlcNAc₂ intoGlcNAc or produce GlcNAc from the nonreducing end of the chitooligomers.In addition, exo-N,N′,N″-acetylchitotriohydrolases cleave monomericunits of GlcNAc from longer chitin chain or release chitotriose which issubsequently cleaved to form diacetylchitobiose and N-acetylglucosamine(Brurberg M. B, Nesl I. F and Eijsink, V. G. H (1996) Comparativestudies of chitinases A and B from Serratia marcescens Microbiol. 142:1581-1589).

Generally, the chitinases known in the art show either endochitinaseactivity or exochitinase activity. The recombinant chitinase enzyme ofthe present invention exhibits the exochitinase activity andendochitinase activity. Fluorogenic assays showed that the recombinant,modified chitinase enzyme of the present invention, BRLA_Chi 90 exhibitstwo types of major exochitinase activity, predominantly a chitobiosidaseactivity and also a prevalent N-acetylglucosaminidase activity, withsome endochitnase activity. Chitinase activity assays using the threetypes of fluorogenic substrates-, 4-MU-β-D-N, N,N″-triacetylchitotriose,4-MU-diacetyl-β-D-chitobioside and 4-MU-N-acetyl-β-D-glucosaminide,showed that the recombinant, modified chitinase enzyme exhibitsendochitinase activity, exo-N,N′-diacetylchitobiohydrolase activity andalso β-N-acetyl-D-glucosaminidase activity.

Zymography analyses and real time ESI-MS studies also confirmed that therecombinant modified chitinase BRLA_Chi 90 is a unique, chitinolyticenzyme with dual enzyme activity, both exo and endochitinase activity.The combined endo- and exo-chitinase activity results in a synergisticincrease in the chitinolytic activity resulting in a greater efficacy ofchitinase for insect control and efficient chitin degradation. Analysisof chitin hydrolysis using colloidal chitin, an insoluble chitinsubstrate, indicates that the recombinant chitinase enzyme can also becapable of producing monomers from longer chitin chain, indicating thatthey also have an endochitinase orexo-N,N′,N″-triacetylchitotriohydrolase activity. While exo- andendochitinases are able to hydrolyze chitin independently to yieldchitooligomers of different length, the presence of both activitiessignificantly enhances the efficiency of chitinolytic machinery. Therecombinant chitinase enzyme of the present invention exhibits both theexochitinase activity and endochitinase activity at a temperature from25° C. to 67° C., a novel and most important desirable feature whichmakes it suitable for various commercial applications in agriculture,medicine and environment.

As demonstrated by most sensitive, reliable ESI-MS experiments, therecombinant, modified chitinase, BRLA_Chi 90 exhibits exo- andendochitinase activity to completely degrade the chitin to yieldchitobiose and N-acetylglucosamine, as major products. The ability ofBRLA_Chi 90 to act on insoluble chitin substrate (colloidal chitin)strongly indicates that it is a “true chitinase” and not achitodextrinase. The recombinant chitinase is suitable for manyindustrial applications, in particular, it could completely utilizenaturally occurring, cheap, abundant sources of chitin wastes from theenvironment like crustacean shells to yield value added chemicals,biofuels and biopolymers.

Though the recombinant, modified chitinase exhibits sequence homologywith two other proteins, chitodextrinase from Brevibacillus laterosporusGI-9 (Genbank Accession No-CCF12514.1) and a chitodextrinase fromBrevibacillus laterosporus LMG 15411 (Genbank AccessionNo-WP_003335299.1), it doesn't exhibit any functional homology withthese proteins.

Chitodextrinase processively hydrolyzes disaccharides from thenon-reducing end of soluble chitin oligosaccharides and is not active onchitin. The chitodextrinase from Brevibacillus laterosporus GI-9 andBrevibacillus laterosporus LMG 15411, is an endo-cleavingchitodextrinase acts only on soluble chitoligosaccharide substrates andit has no activity on insoluble chitin substrates. Irrespective thesource of organism, the enzyme chitodextrinase doesn't solubiliseinsoluble forms of chitin like crystalline chitin (chitin flakes),amorphous chitin (colloidal chitin), it will only act on solublechitooligomers, whereas, a true chitinase will act on native chitin andcompletely degrade it to monomer, N-acetylglucosamine. Two proteins withsimilar sequence homology can be functionally different and havedifferent biological activity and mode of action.

FIG. 7 shows the temperature profile of the recombinant chitinase of thepresent invention. Further, fluorogenic assays revealed that therecombinant chitinase BRLA_Chi 90 of the present invention exhibits anas endochitinase activity and an exochitinase activity(β-1,4-N-acetylglucosaminidase activity and/orN,N′,N″-triacetylchitotriohydrolase activity) at an optimum temperatureof 55° C. and an exochitinase activity,exo-N,N′-diacetylchitobiohydrolase activity) at an optimum temperatureof 60° C.

The optimal pH for the recombinant chitinase BRLA_Chi 90 was determinedby assaying the purified enzyme at different pH (3.0-11.0) usingappropriate buffers and measuring the relative activity under standardassay conditions using fluorogenic 4-methylumbelliferyl substrates. Thedesirable physicochemical features of the recombinant chitinase BRLA_Chi90 includes its activity at a broad range of pH (3.0-11.0) and anoptimum pH-9.0, indicating that it is alkaline active enzyme, adesirable feature suitable for insect control and industrial processingof chitin.

The chitinase enzyme based biological control using novel, chitinasecompositions of the present invention from a new strain ofentomopathogen, Brevibacillus laterosporus Lak 1210, as a potential,contact biopesticide. This aspect of the invention may lead to thedevelopment of a novel, contact insecticidal compositions having therecombinant chitinase, BRLA_Chi 90, as an alternative to Bt-basedbiopesticide, since Bt is effective only when eaten by the insect as alarva. The present invention will majorly focus on its successful use ascontact biopesticide with substantially reduced requirement of frequentapplication, unlike the other biopesticides, which need frequent fieldapplication.

In an embodiment of the present invention, a composition having therecombinant chitinase enzyme of the present invention and a carrier isprovided. Preferred combinations can be formulated with an acceptablecarrier into a biopesticide/biocontrol agent. Furthermore, thesecombinations of this invention have an advantage of being formulated asan adjuvant, a colloid, a wettable powder, an emulsifiable concentrate,an aerosol or spray, a dusting powder, a dispersible granule or pellet,an impregnated granule, a suspension, a solution, an emulsion and alsomicroencapsulations. The compositions can be formulated by conventionalmethods such as those described in, for example, Winnacker-Kuchler(1986), “Chemische Technologie” [Chemical Technology], Vol. 7, C. MauserVerlag Munich, 4th Ed. 1986; van Valkenburg, “Pesticide formulations”,Marcel Dekker N.Y., 2nd Edition 1972-73; K Martens, “Spray DryingHandbook”, 3rd Edition, G. Goodwin Ltd. London. Necessary formulationaids include carriers, inert materials, surfactants, solvents, and otheradditives. Such formulated compositions may be prepared by concentrationof a culture of cells having the polypeptides by methods likedesiccation, extraction, filtration, centrifugation, sedimentation,homogenization and lyophilization.

The composition of the present invention can be a contact biopesticide,ingestion biopesticide, biofungicide, bioinsecticide, or abionematicide. In certain exemplary embodiments, insecticidal andantifungal compositions for enzyme-based formulations containing therecombinant, modified chitinase, BRLA_Chi 90 are provided. Specificembodiments also provide screening of different classes of compositionsor formulations based on the determined and desired characteristics ofthe recombinant, modified chitinolytic enzyme, BRLA_Chi 90. Theantifungal and insecticidal compositions can include culture supernatantcontaining the secreted recombinant, modified chitinase, BRLA_Chi 90and/or culture broth containing the whole cells of recombinant E. colicarrying the expression vector pET/BRLA_Chi90. The insecticidal andantifungal compositions can be formulated as dry formulations (wettablepowders, dry flowables, dust, granules) or b) liquid formulations(oil-based, aqueous- based) or combinations thereof. For liquidcompositions, the antifungal and insecticidal compositions includingrecombinant, modified chitinase, BRLA_Chi 90 can be blended with mineralor vegetable-based oil carrier and emulsifiers or stabilizers, stickers,surfactants and antifreeze compounds. Dry compositions or formulationscan include an inert carrier (peat, vermiculite etc) or chitin andchitosan materials or natural chitinous sources like crustacean shells.The antifungal and insecticidal compositions can also be formulated ascontrolled release formulations (nanoformulations based on nanomaterialsor microencapsulated formulations using microencapsulationtechnologies).

The present invention is also directed to a method of protecting ortreating or modulating phytopathogenic infection in a plant or a partthereof including applying a composition as disclosed. Thephytopathogenic infection in a plant can be caused by a fungi or aninsect. An effective amount of the compositions of the present inventioncan be applied to the environment hosting the target insectpest/pathogenic fungus, e.g., soil, water or a plant or a part thereofwherein seeds, plantules, plants, foliage of plants of the plant totreat the infection.

More specifically, exemplary embodiments provided methods for utilizingnucleotide sequences and their encoding polypeptides with insecticidaland antifungal activity produced by the recombinant microorganisms andalso methods and compositions of chitinase enzyme-based formulations forimpacting insect pests and phytopathogenic fungi.

The phytopathogenic infection as described in the present invention is aplant disease caused by at least one fungus selected from the groupconsisting of Fusarium, Rhizoctonia, Pythium, Phytophthora, Cercospora,Puccinia, Venturia, Alternaria, Uncinula, Ustilago, Colletotrichum,Erysiphe, Botrytis, Sclerotium and Monihnia which comprises contactingsuch fungus with purified antifungal chitinases effective to obtain saidinhibiting. The method of the present invention involving application ofa chitinolytic enzyme can be carried out through a variety of procedureswhen all or part of the plant is treated, including seeds, roots, stemsand leaves etc.

The examples of the Fusarium species include Fusarium oxysporum,Fusarium lycopersici, Fusarium moniliforme, Fusarium graminearum andFusarium equiseti.

The active compounds and compositions of the embodiments displayactivity against insect pests, which may include economically importantagronomic, forest, greenhouse, nursery, ornamentals, food and fiber,public and animal health, domestic and commercial structure, household,and stored product pests. Insect pests include insects selected from theorders Diptera, Lepidoptera, Coleoptera, Hymenoptera, Homoptera,Hemiptera, Orthoptera, Thysanoptera, Isoptera, more specifically insectsfrom Diptera, Coleoptera and Lepidoptera.

Further, the phytopathogenic infection as described in the presentinvention may be caused by at least one insect belonging to Lepidoptera,Diptera, Coleoptera, Homoptera or Hymenoptera.

In a preferred embodiment of the present invention there is provided amethod for protecting or treating or modulating phytopathogenicinfection in plant or a part thereof, wherein the method comprisesapplying the composition as disclosed in the present invention as acontact biopesticide. In another embodiment, there is provided a methodfor protecting or treating or modulating phytopathogenic infection inplant or a part thereof, wherein the method comprises applying thecomposition as disclosed in the present invention as an ingestionbiopesticide in an effective amount. The ingestion biopesticide can beapplied in an effective amount to damage the midgut in said insectlarvae and insects.

In another embodiment of the present invention there is provided amethod for protecting or treating or modulating phytopathogenicinfection in plant or a part thereof, wherein the method comprisesapplying the composition as disclosed in the present inventionconcurrently with the Bacillus-based insecticides, wherein the methodenhances the insecticidal effectiveness of the Bacillus-basedinsecticides for insect control.

In an embodiment, the present invention provides a safe, biopesticidalcomposition including culture supernatant containing the recombinantchitinase enzyme and/or cell suspensions of recombinant E. colisecreting the recombinant chitinase enzyme, as a contact and ingestion(stomach) biopesticide. In an embodiment, toxicological studies wereperformed to prove the safety in application of biopesticidalcompositions of the present invention as a contact and ingestionbiopesticide (Example 16). The recombinant, modified chitinase, BRLA_Chi90 purported to be used on agricultural fields, horticulture farms,forests, forest nurseries is evaluated for interim risk assessmenttowards non-target animals.

Low or non-existing toxicity of the culture supernatant containing therecombinant chitinase enzyme and/or cell suspensions of recombinant E.coli secreting the recombinant chitinase enzyme, through three differenttoxicity studies in animal models, such as acute oral toxicity in amurine model (adult Wistar rats) and a dermal irritation test inrabbits. The preliminary toxicological profiling of the recombinantchitinase enzyme provided useful and necessary information for riskassessment and indicates an absence of toxicity and pathogenicity of thechitinase to mammals in laboratory animals (rats and rabbits), guidingthe choice for development of a “low risk” next generation, potentialbiopesticide, as a possible alternative to Bt.

In an aspect, the compositions including the recombinant chitinase ofthe present invention can be used in combination with other Bt-basedbiopesticides or other proteins with insecticidal activity to increaseinsect target range for insect resistance management and control.Furthermore, the compositions can also be used in combination with otherbiocontrol agents or chemical fungicides for integrated pest management.

The use of recombinant chitinase as an ingestion biopesticide, as analternative to Bt-based biopesticide, with substantially reduced threatof resistance to be developed. The biological efficacy of therecombinant chitinase, modified BRLA_Chi 90, of the present inventionmakes it an attractive bioinsecticide/ biocontrol agent/ and abiofungicide. The development of chitinase-enzyme based technology basedon recombinant chitinase, BRLA_Chi 90 from Brevibacillus laterosporus asa next generation biopesticide and possible alternative to Btcontributes to an innovation in biopesticide research. The chitinaseenzyme-based biological control using Brevibacillus laterosporus Lak1210 could provide a preferred solution for integrated pest management,especially for the control of major insect pests and fungal diseases ofsignificance to forestry and agriculture.

The special characteristics of enzymes exploited for their commercialinterest and industrial applications include thermotolerance, toleranceto a varied range of pH, stability of enzyme activity over a range oftemperature and pH, and other harsh processing conditions. Therecombinant, modified chitinase of the present invention possess manyattributes required for an ideal biopesticide for use in integrated pestmanagement programs and with possible applications in bioremediation andbiofuel industry. The recombinant chitinase functions in a broad pHrange (pH 3.0-11.0), highly alkaline active with a pH optimum of 9.0,thermoactive with a temperature optimum of 55-60° C. and hence could beused in several industrial bioprocesses followed by scale up andcommercialization.

In a further embodiment, using the method of chitin affinity adsorptionthe recombinant, modified chitinase BRLA_Chi 90 secreted into theculture medium can be adsorbed on to crustacean shells for theutilization of fishery wastes generated from seafood processingindustries. It can be proposed to be not only as an efficient,inexpensive method of bioremediation for the disposal of marine foodwastes (crustacean shells like crab shells, shrimp shells and krillshells) contributing to a substantially cleaner, greener environment butpossibly an environmentally benign alternative to extract industrialchitin and chitosan over the traditional chemical method of extraction.

Using the method of chitin hydrolysis herein described, chitin affinityadsorption method is also used for reclamation of the crustacean shellsfor the production of industrially important chitooligomers,N-acetylglucosamine and glucosamine of therapeutic interest.

The recombinant chitinase of the present invention, BRLA_Chi 90 is anunique enzyme which exhibits endochitinase activity, and two types ofexochitinase activity. The desirable physicochemical features of therecombinant, modified chitinase, BRLA_Chi 90 includes, its activity atvaried pH (pH 3.0-11.0), alkaline activity (optimum pH-9.0) andthermoactivity (optimum temperature-55-60° C.), inherent thermostability(Tm of 66.7° C.) and specificity towards insoluble substrates, inparticular natural substrates like chitin rich crustacean shells, whichare some of the key considerations for the proposed application ofrecombinant, modified chitinase, BRLA_Chi 90, in a diverse spectrum ofindustrial processes.

The recombinant chitinase of the present invention produces industriallyimportant chitobiose and N-acetylglucosamine as major end products, insubstantial amounts. The recombinant chitinase is a robust enzyme withindustrial applicability because of its unique chitin degradationability, activity, and stability, since the industrial use of thechitobiose and N-acetyl glucosamine have been limited by the lack ofenzyme efficient biotechnological process to produce the large scaleproduction of these commercially valuable products.

In an aspect, the combined endo- and exo-chitinase activity results in asynergistic increase in the chitinolytic activity resulting in greaterefficacy of chitinase for insect control and chitin hydrolysis.Evaluation of the efficacy and effectiveness of the recombinantchitinase of the present invention is shown in Examples 1 to 18.

EXAMPLES

Following are the illustrative and non-limiting examples, including thebest mode, for practicing the present invention.

Example 1 Genomic DNA Extraction and PCR Amplification of ChitinaseGene, B/Chi

PCR amplification of B/Chi gene from Brevibacillus laterosporus Lak 1210(FIG. 1)

Genomic DNA was extracted from Brevibacillus laterosporus Lak1210 (MTCC5487) as described by Pospiech and Neumann. Briefly, cells ofBrevibacillus laterosporus were grown in Nutrient Broth with 1%colloidal chitin at 30 C and 200 rpm. Cells were pelleted down andresuspended in 0.5 ml of saline-EDTA (150 mM NaCl, 100 mM EDTA)containing 30 mg/ml lysozyme and 40 mg/ml of RNase. Following incubationat 65° C. for 15 min, proteinase K was added to a concentration of 200mg/ml followed by 10 ml of a 25% sodium dodecyl sulfate (SDS) solution.After incubating at 65° C. for 15 min, the resulting lysate was chilledbriefly on ice and then extracted with phenol-chloroform method.

The extracted genomic DNA was used as a template to amplify chitinasegene. BLASTX analysis (www.ncbi.nlm.nih.gov/blast) was performed forselecting the closest chitinase genes and the assembled candidatechitinase sequences were subjected to contig analysis with the DNAMANsoftware (Version 5.2.2, Lynnon BioSoft, Quebec, Canada) to design theprimers. All oligonucleotide primers used in this study as listed inTable 2, were designed using the software PRIMER 3 (Frodo.wi.mit.edu/).A pair of gene specific primers chi F(5′-TAACAACAATGATATGAACTGACCTAAG3′) and Chi R(5′-CTTCTTCATTTCCAAACGCAGTCATA3′) was used to amplify the full lengthopen reading frame (ORF) of 2583 bp. Genomic DNA was digested with HindIII and chitinase gene was amplified in a reaction volume of 25 μLreaction having 50 ng genomic DNA, 50 mM each of dNTPs, 1.5 μl DMSO and100 ng of each primer, 5× GC buffer and 0.5 units of q5 DNA polymerase.

TABLE 2 Oligonucleotide primers used in this study OligonucleotideSequence (5′-3′) Length Tm Description CHI-F TAACAACAATGATATGAACT 28-mer54.1° C. Primer for GACCTAAG amplification of chi ORF from genomic DNA,forward CHI-R CTTCTTCATTTCCAAACGCA 26-mer 54.8° C. Primer for GTCATAamplification of chi ORF from genomic DNA, reverse CHI-1 IFCTTATTGCCAAGCACATGAT 30-mer 61.6° C. Primer for site- CAGGATGGACdirected mutagenesis, internal forward 1 CHI-1 IR GTCCATCCTGATCATGTGCT30-mer 60.1° C. Primer for site- TGGCAATAAG directed mutagenesis,internal reverse 1 CHI-2 IF GCAGCAACCCCACATGATGC 29-mer 61.5° C.Primer for site- TTCAAATAG directed mutagenesis, internal forward 2CHI-2 IR CTATTTGAAGCATCATGTGG 29-mer 61.5° C. Primer for site- GGTTGCTGCdirected mutagenesis, internal reverse 2 CHI_F GCGGCGCATATGAGAAAGA41-mer 57.3° C. Cloning TGTATCAACACATTCCTACT GC^(a) CHI_RGCCGCCCTCGAGCTTCTTCA 40-mer   57° C. Cloning TTTCCAAACGCAGTCATATC^(b)^(a)The underlined nucleotide sequence is Nde 1 restriction site ^(b)Theunderlined nucleotide sequence is Xho 1 restriction site The singlebases underlined are mutated bases from the native chitinase sequenceshown in bold face

All the PCR reactions were performed by in the thermal cycler (Bio-Rad,Hercules, Calif., USA) with standard high fidelity phusion PCR protocolusing high fidelity polymerase, Q 5 phusion polymerase (New EnglandBiolabs, USA) using the following cycling parameters: 30 s initialdenaturation at 98° C., followed by 35 cycles of 10 s at 98° C., 20 sannealing at 60° C., and 60 s at 72° C. followed by a final extension of10 min at 72° C. (Table 3) .

TABLE 3 PCR reaction conditions for amplification of B/chi gene StepTemperature Time Initial denaturation 98° C. 60 s 35 cycles 98° C. 10 s60° C. 20 s 72° C. 60 s Final extension 72° C. 10 min

The amplified PCR product was sized by electrophoresis in 1% agarosegels, purified using quick Clean DNA gel extraction kit (Qiagen,Netherlands). The nucleotide sequence of the amplicon was confirmedusing a BigDyeTerminator cycle sequencing kit and an automated DNAsequencer (Applied Biosystems, Calif., USA).

The amplified PCR product is 2583 bp in length and designated as B/Chishown in FIG. 1. The sequence of native chitinase gene is listed asSEQUENCE ID No:1.

Example 2 Site Directed Mutagenesis, Cloning and Construction ofExpression Plasmid pET/BRLA_Chi 90

Site directed mutagenesis was carried out by overlapping extension-PCRusing pET/BRLA_Chi 90 expression plasmid as a template. A single pointmutation were introduced to the full length ORF (2583 bp) that waspreviously cloned into the pET 21 b expression vector to modify the genesequence using mutagenic primers to generate a restriction site suitablefor Nde 1 and xho 1.

The forward mutagenic primers used for site directed mutagenesis are:

5′-CTTATTGCCAAGCACATGATCAGGATGGAC-3′ and GCAGCAACCCCACATGATGCTTCAAATAG.

The reverse sequence is 5′-GTCCATCCTGATCATGTGCTTGGCAATAAG andCTATTTGAAGCATCATGTGGGGTTGCTGC-3′.

The nucleotide underlined represents the mutated nucleotide and thesequences underlined CACATG and CATGTG represents the restriction sitefor the enzymes Nde I and xho I, respectively. The mutant clones wereselected after sequencing the entire open reading frame to ensure thatthe desired mutation was successfully introduced as the only mutation inthe mutated gene. The mutated sequence of the chitinase gene BlChi waslisted as SEQUENCE ID No: 3.

The amplicon BlChi (2583 bp) was digested with Nde 1 and Xho 1 andcloned into the pET-21 b (+) vector (Novagen), resulting in theconstruction of recombinant expression plasmid pET/BRLA_Chi 90. Therecombinant expression vector was constructed such that the nativesignal peptide sequence of Brevibacillus laterosporus LAK 1210 was inframe with the C-terminus. The expression vector pET/BRLA_Chi 90 wastransformed into E. coli BL21 (DE3) competent cells.

Recombinants selected on LB agar plates containing 100 μg/ml ampicillinwere analyzed by colony per. Six positive genomic clones carrying thefull length ORF (2583 bp) were chosen and the recombinant clones wereverified by sequencing using gene specific (CHI-F and CHI-R) and vectorspecific (T7 forward and T 7 reverse) primers. The sequence of the DNAinsert was confirmed using 3730XL DNA Analyzer (Applied Biosystems, CA,USA).

The ORF (2583 bp) translates to a polypeptide of 860 amino acid residueswith a calculated molecular mass of 89.68 kDa and pI of 5·93. Thededuced chitinase enzyme was designated as BRLA_Chi90. The deducednative and mutated amino acid sequence of the recombinant BRLA_Chi 90was listed as SEQUENCE ID NO: 3 and SEQUENCE ID No: 4 respectively. Themutated amino acid sequence of the recombinant BRLA_Chi 90, withoutnative signal sequence was listed as SEQUENCE ID NO: 5. The nativesignal peptide sequence with 42 amino acids was listed as SEQUENCE IDNO: 40. The nucleotide sequences and their encoded sequences werealigned by running a BLAST search on NCBI (blast.ncbi.nlm.nih.gov/blast.cgi) and analyzed the DNAMAN software package(Version 5.2.2, Lynnon BioSoft, Vaudreuil Dorton, Quebec, Canada). Thenucleotide sequences for the native chitinase and the recombinant(mutated) chitinase sequence were deposited with the GenBank with theaccession numbers MF397932 and MF 397933, respectively.

Example 3 Expression, Large Scale Production and Purification ofRecombinant, Modified Chitinase, BRLA_Chi 90

SDS-PAGE analysis of recombinant, modified chitinase, BRLA_Chi 90 (FIG.2A)

For protein expression, large scale production and purification,positive clones of Escherichia E. coli BL21(DE3) harboring thepET/BRLA_Chi 90 vector were grown in Terrific Broth medium supplementedwith 100 μg/ml ampicillin at 37° C. to OD₆₀₀ nm of 1.0. Expression ofpET/BRLA_Chi 90 was induced with 0.1 mM IPTG and the cells were grownfor different time intervals to optimize the overexpression andextracellular secretion of the recombinant chitinase. To analyzeexpression levels of the secretory protein and the optimalpost-induction time for harvesting the cells, the culture supernatantwas collected at 1, 2, 3, 6, 8 and 12 h and the protein samples wereanalyzed by 12% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). The cells were harvested after 6 h,post-induction by centrifugation at 8000g, 4° C., for 10 min, washedwith STE buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 300 mM NaCl) and weresonicated on ice (5 cycles, 30 s, each).

The recombinant chitinase was purified from the concentrated culturesupernatant and the cytoplasmic fraction of the cell lysate by a singlestep chitin affinity chromatography using chitin beads (New EnglandBiolabs, USA). The cell lysate/ammonium sulphate dialysate /concentratedculture supernatant was loaded onto a 2 ml Poly-Prep chromatographycolumns (9×0.8×4 cm) (Amersham Biosciences, USA) packed with chitinbeads (New England Biolabs, USA) previously equilibrated with 20 mM TrisHCl (8.0) and the bound proteins were eluted with 20 mM Tris HCl (8.0)and the recombinant chitinase was eluted stepwise with 0.5 M, 0.1 M and20 mM acetic acid. Fractions with high chitinase activity were collectedand concentrated using ultrafiltration. The yield for the recombinant,modified chitinase is about 6 mg/L and could further improved byoptimizing the process conditions.

A cost-effective, large scale purification method was developed bychitin affinity adsorption chromatography to selectively adsorb theextracellular chitinase present in the culture supernatant/fermentationbroth. The culture supernatant was concentrated using AMICON PM 30 (MWCO30 kDa). Borosil glass chromatography columns (500 mm) fitted with astopcock were packed with pretreated, powdered crustacean shells (crabshells or, shrimp shells). The concentrated culture supernatant wasloaded onto powdered crustacean shell matrix, previously equilibratedwith 20 mM Tris HCl (8.0) and the bound proteins were eluted with 20 mMTris HCl (8.0). After several washes with distilled water, therecombinant chitinase was eluted stepwise with 0.5 M, 0.1 M and 20 mMacetic acid.

Powdered, crude shrimp shell chitin exhibited an adsorption capacity of95.2 U/g which was 36.3% higher than the powdered, crude crab shellchitin. The recombinant chitinase, when eluted with 20 mM acetic acidresulted in 93% chitinase recovery with a purification fold of 9.6. Theenhanced adsorption could be due to the efficient binding of thesubstrate binding domain of BRLA_Chi 90 to the insoluble chitinsubstrates chitin hydrolysis and also efficient chitin hydrolysis due tothe unique dual enzyme activity.

The purity of BRLA_Chi 90 was analyzed using 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecularmass (94488 Da) calculated from the deduced amino acid sequence withoutthe signal peptide is in reasonable agreement with the molecular mass(89680 Da) of the recombinant chitinase assessed by SDS-PAGE (FIG. 2 A)indicating that BRLA_Chi 90 is a monomeric enzyme. The aminoacidsequence of the recombinant chitinase expressed as a mature protein(without signal peptide) was listed as SEQUENCE ID NO: 5.

Example 4 Zymography Assays (In-Gel Activity Staining) UsingRecombinant, Modified Chitinase BRLA_Chi 90

Chitinase zymography with 4-methylumbelliferyl substrates usingrecombinant, modified chitinase BRLA_Chi 90 (FIG. 2B)

In gel activity staining assays were performed for rapid detection andsemi-quantitative analysis of chitinase by using agarose overlaycontaining fluorogenic chitooligosaccharide analogs,4-methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MU-GlcNAc),4-methylumbelleferyl N,N′-diacetyl-β-D-chitobioside (4-MU-GlcNAc₂) and4-methylumbelleferyl N,N′,N″-triacetyl-β-D-chitotrioside (4-MU-GlcNAc₃)(Sigma Chemicals, St Louis, USA) for N-acetyl-beta-glucosaminidase,chitobiosidase and endochitinase, respectively.

Protein samples were separated on 12% SDS-PAGE. After electrophoreticseparation, the recombinant enzyme was renatured by incubating the gelin 0.1M sodium acetate buffer (pH 5.0) containing 1% (v/v) Triton X-100at 37° C. for 2 h. An overlay gel containing 4-methylumbelliferylsubstrate (10 μm in 1% agarose) was overlaid on the slab gel and the gelsandwich was allowed to incubate at 37° C. for 15 min. The enzymeactivity was visualized as bright, fluorescent bands where enzyme hadreleased 4-methylumbelliferone from the chitin analog.

Zymography analyses revealed that the recombinant chitinase exhibited ingel activity against all the three fluorogenic chitooligosaccharideanalogs indicating that it has a combined exo- and endochitinaseactivity (FIG. 2B).

Example 5 Secretion Profile, Extracellular Targeting of the Recombinant,modified Chitinase BRLA_Chi 90 and Evaluation of Membrane Integrity(Cell Leakage/Permeability/Viability) by β-Galactosidase Activity

Secretion profile of recombinany, modified chitinase BRLA_Chi 90expressed in the different cell compartments (FIG. 3)

Cells were harvested by centrifugation at 5,000 g for 20 min at 4° C.,and the supernatants were pooled (culture medium with extracellularfraction). Cells were fractionated to separate the recombinant proteinsinto periplasmic, cytoplasmic and with the EDTA/lysozyme/cold osmoticshock method. Cells were suspended in 30 mL of 20 mM Tris-HCl (pH 7.4)containing 20% sucrose (w/v), 0.5 mM EDTA and a protease inhibitor,incubated on ice for 30 min, and then centrifuged at 15,000 g for 15 minat 4° C. After the addition of 120 mL of 20 mM Tris-HCl (pH 7.4)containing the protease inhibitor, the cells were further incubated onice for 30 min and centrifuged at 15,000 g for 15 min at 4° C. Thesupernatants were combined (periplasmic fraction). The resulting cellpellet was suspended in STE buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA,300 mM NaCl) and sonicated and centrifuged at 15,000 g for 15 min at 4°C. and the supernatant was pooled (cytoplasmic soluble fraction).

The BlChi gene ORF with its native signal peptide was efficientlysecreted, at first into the periplasmic space of E. coli cells and thenexcreted into the culture medium. Targeting of the recombinant chitinaseBRLA_Chi 90 into the extracellular medium allows efficient purificationby chitin adsorption chromatography using shrimp shell and crab shellchitin.

f3 Galactosidase is a cytosolic protein and the extracellularβ-galactosidase activity detected in the culture medium was used as anindicator of cell leakage/lysis. Post-induction, the culture supernatantwas collected at different time points and assayed for β-galactosidaseactivity. The β-galactosidase activity was quantified by determining theamount of β-galactosidase in the extracellular medium usingo-nitrophenyl-D-galactopyranoside (ONPG). The enzyme assay mixturecontains 50 μl of the substrate buffer containing 2.0 mg/ml ONPG in 0.2M phosphate buffer (pH 7.2) was added to 50 μl of the sample, which wasthen incubated at 37° C. for 10 min. The reaction was stopped byaddition of 0.1 ml of 1 M sodium carbonate, and the absorbance was readat 420 nm. One unit of β-galactosidase is defined as the amount whichhydrolyzes 1 μmol of ONPG to o-nitrophenol and D-galactose per minuteunder the experimental conditions.

We have observed that the amount of β-galactosidase activity in theculture medium concomitantly increased with increase in chitinaseactivity from 1 h to 24 h (FIG. 3). These results demonstrated that theimproved secretion during the cultivation period is independent of celllysis and due to the possible membrane permeabilization/leakage of E.coli cells.

Example 6 In Silico Analysis of the Recombinant, Modified Chitinase,BRLA_Chi 90

Multiple sequence alignment comparison of the recombinant, modifiedchitinase, BRLA_Chi 90 of Brevibacillus laterosporus with the conserveddomains of chitinases from entomopathogens (FIG. 4)

Multiple sequence alignment comparison of the recombinant, modifiedchitinase, BRLA_Chi 90 of Brevibacillus laterosporus with the conserveddomains of chitinases from other bacterial chitinases (FIG. 5)

Structural Analysis of the recombinant, modified chitinase atsubstitution site through protein structure prediction approach (FIG.30)

The identification of nucleotide sequences from the chitinaserecombinant clones was established using the NCBI Blast program(www.ncbi.nlm.nih.gov/BLAST). Open reading frame and protein predictionwere made using NCBI ORF Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html).Sequence alignment was conducted and a phylogenetic tree was constructedusing the DNAMAN software ((Version 5.2.2, Lynnon BioSoft, Quebec,Canada).

Multiple sequences alignment of BRLA_Chi 90 and other chitinasesequences retrieved from the predicted proteomes of the genomes fromNCBI (www.ncbi.nlm.nih.gov) was done more importantly by identifying theconserved active site signature motif DXXDXDXE. Comparison of thepredicted BRLA_Chi 90 chitinase sequence with chitinase sequences fromentomopathogens and other bacterial chitinases. It revealed two highlyconserved consensus motifs (SIGG and EGIDIDYE) corresponding to asubstrate-binding site and catalytic domain respectively confirmed thatBRLA_Chi 90 was a member of glycosyl hydrolase family 18 (FIGS. 4 and5).

The consensus sequence ((EGIDIDYE) corresponding to the catalytic domainof BRLA_Chi 90 is novel and different from the other chitinases. Thenovelty of the consensus sequence of the catalytic domain attributes tothe unique functional properties of BRLA chi 90.

The sequence of BRLA_Chi 90 was analyzed to evaluate their theoreticalisoelectric point (pI) molecular weight percentage of amino acidcomposition (%)), number of positively and negatively charged residues,extinction coefficient, instability and aliphatic index, Grand Averageof Hydropathy (GRAVY) using Expasy Protparam tool(us.expasy.org/tools/protparam.html). Protein sequences encompassingsignal peptides and the putative signal peptide cleavage site waspredicted by SignalP and TargetP tools (www.cbs.dtu.dk/services/SignalP/and www.cbs.dtu.dk/services/TargetP/).

Subcellular localization presumption was performed using ProtComp andWOLF PSORT to refine the secretome predictions.(linux1.softberry.com/berry.phtml?topic=protcomppl&group=programs&subgroup=prolocand wolfpsort.org).

Secondary structure was predicted using PSIPRED server(bioinfcs.ucl.ac.uk/psipred/). The three-dimensional structure of therecombinant chitinase BRLA_Chi 90 protein domain was predicted using theSWISS-MODEL workspace (swissmodel.expasy.org) using a chitinase templateof chitinase from Chromobacterium violaceum ATCC 12472 (GenBankAccession No. GI:939186282; PDB code 4 TXG_A).

The ORF (2583 bp) translates to a gene product of 860 amino acidresidues with a deduced molecular mass of 94.4 kDa and a pI of 5.93. Themolecular mass of the mature chitinase (89.68 kDa) as estimated byMALDI-TOF and SDS-PAGE analyses which corresponds well with the deducedmolecular mass (94.4 kDa). The signal peptide prediction by both SignalP v4.1 (www.cbs.dtu.dk/services/SignalP/) and Target P v1.1(www.cbs.dtu.dk/services/TargetP/) predicted that BRLA_Chi 90 containeda putative N-terminal signal peptide of 42 amino acids (SEQUENCE ID:40). The predicted signal sequence of 42 amino acids at the N-terminusof the deduced BRLA_Chi 90 exhibited a typical feature of the signalpeptide characteristic of Grampositive bacteria producing secretedproteins. The recombinant chitinase, BRLA_Chi 90 was predicted to becleaved by a signal peptidase between positions A 41 and S 42.

Soluble proteins tend to have polar or hydrophilic residues on theirsurfaces. The site directed mutagenesis at positions 661 and 2158 in thenative chitinase gene resulted in the aminoacid substitution at position221 and replacing tyrosine with histidine. The substitution of a polar,hydrophilic histidine (H) residue for a hydrophobic aromatic aminoacidtyrosine (Y) h may increase the interaction with the negatively chargedinsect cuticle. The substitution also resulted in improved solubility ofthe protein, another important desirable characteristic feature suitablefor biocontrol and other industrial applications of chitinase.

The structure-function of the modified chitinase at the substitutionsite was predicted using Rosetta (version 3.4) software. The predicted3D structure of the recombinant, modified chitinase showing surfaceexposed histidine at position 221 as depicted in FIG. 30.

Example 7 Mass Analysis and Peptide Mass Fingerprinting of theRecombinant, Modified BRLA_Chi 90

MALDI-TOF/MS spectrum of purified BRLA_Chi 90 (FIG. 6)

Listing of unique peptides from peptide mass fingerprinting ofrecombinant, modified chitinase, BRLA_Chi 90 (SEQUENCE ID NO: 7-SEQUENCEID NO: 39) Table 1

Recombinant chitinase, BRLA_Chi 90 was applied in parallel onto a 12%SDS-PAGE gel using a Laemmli buffer system. Following electrophoresis,protein was stained with Coomassie blue. After destaining, protein bandswere excised from the gel and in-gel digested with trypsin (sequencinggrade, Promega, Wis., USA) using a standard protocol. After overnightdigestion at 37° C., the peptides were extracted, dried in a SpeedVacvacuum centrifuge and resuspended in 10 μl of 70% acetonitrile.MALDI-TOF-MS using the UltrafleXtreme MALDI-TOF/TOF (tandem TOF) system(Bruker Daltonics) and α-Cyano-4-hydroxycinnamic acid (5 mg/ml in 60%acetonitrile in 1 mM citric acid) was used as MALDI matrix. An aliquot(1 μl) of the extracted supernatant was mixed with 1 μl of matrixsolution directly on the MALDI target plate. Measurements were done inthe positive reflectron mode.

For peptide mass fingerprinting, all experiments were performed on aDionex Ultimate 3000 nano-LC system (Sunnyvale Calif., USA) connected toa linear ion trap-Orbitrap (LTQ-Orbitrap) mass spectrometer(ThermoElectron, Bremen, Germany) equipped with a nanoelectrospray ionsource. The mass spectrometer was operated in the data dependent mode toautomatically switch between OrbitrapMS and LTQ-MS/MS acquisition.Survey full scan MS spectra (from m/z 300 to 2000) were acquired in theOrbitrap with resolution R=60,000 at m/z 400 (after accumulation to atarget of 1,000,000 charges in the LTQ). The method allowed sequentialisolation of the most intense ions, up to six, depending on signalintensity, for fragmentation on the linear ion trap using collisionallyinduced dissociation at a target value of 10,000 charges. General massspectrometry conditions were: electrospray voltage, 1.5 kV; no sheathand auxiliary gas flow. Ion selection threshold was 500 counts forMS/MS, and an activation Q-value of 0.25 and activation time of 30 mswere also applied for MS/MS.

MS/MS peak lists from individual RAW files were generated using DTASuperCharger package, version 1.29, available at the MSQuant validationtool. Protein identification was performed by using MASCOT Deamon formultiple searches submission on a local Mascot server v2.1 (MatrixScience) The fragment spectra obtained by tryptic digestion wereevaluated and submitted to the bacteria subset of the chitinasedatabase. The search parameters used were: Enzyme: Trypsin/P (no prolinerestriction); Maximum missed cleavages: 3; Carbamidomethyl (C) as fixedmodification; N-acetyl (Protein), Oxidation (M), pyro-glu (Q) andpyro-glu (E) as variable modifications; Peptide mass tolerance of ±15ppm; MS/MS mass tolerance of 0.5 Da. Under these criteria, Mascotindicated a minimal score of 22 for p≤0.01 and 15 for p≤0.05. All datahad an average mass accuracy of 2.8 ppm. Spectra and protein validationwere performed using an open source software called MSQuant (version1.5a61), largely used for LC-MS/MS data analysis. Proteins werevalidated statistically, based on the score of their individualpeptides. Tryptic peptides with a minimal score of 22 for each (proteinfalse-positive probability of 0.01%) but a MS/MS score higher than 38were accepted (protein false-positive probability lower than 0.25%).Using these criteria, all MS/MS identifications of peptides present inentries with reversed sequences (i.e. false positive identifications)were not validated, since none of the Identifications with only oneunique peptide were accepted only after manual validation. Qualitycriteria for manual validation were the assignment of major peaks, theoccurrence of uninterrupted y- or b-ion series of at least 3 consecutiveamino acids, the preferred cleavages N- terminal to proline bonds andC-terminal to Asp or Glu bonds, and the possible presence of a2/b2 ionpairs.

The identified peptide sequences are novel compared to the previouslyreported sequences. The spectra matched 33 tryptic peptides (SEQUENCE IDNO 7-39) (Table 1) that could be correlated to the peptide sequencesfrom chitinase (Uniprot KB-A0A0F7C0B6_BRELA) from Brevibacilluslaterosporus ATCC 64 and chitodextrinase (UniprotKB-A0A075R004_BRELA)from Brevibacillus laterosporus LMG 15441 with 100% identity.

Though recombinant chitinase, Chi 90 shows 99% identity withChitodextrinase (CCF12514.1) from Brevibacillus laterosporus (GI 9),they don't share any functional homology. Irrespective the source oforganism, the enzyme chitodextrinase doesn't solubilise insoluble formsof chitin like crystalline chitin (chitin flakes), amorphous chitin(colloidal chitin), it will only act on soluble chitooligomers.(www.prospecbio.com/Chitodextrinase_8_50/ andwww.uniprot.org/uniprot/P96156). Whereas, a true chitinase will act onnative chitin and completely degrade it to monomer, N-acetylglucosamine.

As demonstrated by most sensitive, reliable ESI-MS experiments, therecombinant, modified chitinase, BRLA_Chi 90 is predominantly anexochitinase (with chitobiosidase activity) acting on insoluble,colloidal chitin to yield chitobiose which is then converted to monomer,N-acetylglucosamine. The ability of Chi 90 to act on native chitin(insoluble colloidal chitin) strongly indicates that it is “truechitinase” and not a chitodextrinase. The complete degradation of nativechitin also demonstrates that Chi 90 also exhibits N-acetyl μglucosaminidase activity proving that it is an efficient chitinasesuitable for industrial applications, also taken into consideration, itsother novel characteristics (FIGS. 12-18, 19A-19C).

The examples clearly illustrate that the recombinant modified chitinaseis a novel enzyme since two proteins with similar sequence homology canbe functionally different and have different biological activity andmode of action.

Example 8 Determination of Chitinase Activity by Fluorogenic Assays,Optimum, pH and Temperature Profile of Recombinant, Modified ChitinaseBRLA_Chi 90

Optimum pH profile for exo- and endochitinase activity using fluorogenic4-methylumbelleferyl substrates (FIG. 7)

Optimum temperature profile for exo- and endo chitinase activity usingfluorogenic 4-methylumbelleferyl substrates (FIG. 8)

Most sensitive and reliable microplate fluorometric enzyme assays wereperformed using fluorogenic 4-methylumbelleferyl substrates in a Enspiremultimode plate reader (Perkin Elmer Inc, Japan) at an excitationwavelength of 360 nm and an emission wavelength of 450 nm. Chitinolyticactivity was fluorimetrically assayed by using fluorogenic chitinanalogs, 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (4-MU-GlcNAc₁),4-methylumbelliferyl N, N′-diacetyl-β-D-chitobioside (4-MU-GlcNAc₂) and4-methylumbelliferyl N,N′,N″-triacetyl-β-D-chitotrioside (4-MU-GlcNAc₃)as substrates to detect exochitinase and endochitinase activity.Chitinase specificity was estimated by the cleavage of the β-1,4-bondthat releases 4-methyllumbelliferone from the different fluorogenicsubstrates.

In a standard assay, a mixture of 10 μM of 4-MU substrate in 0.05 Msodium acetate buffer (pH 5.0) and 1 μL (0.1 μg) of purified enzyme in atotal volume of 100 μL was incubated at 37° C. for 5 min as describedpreviously (56). The reaction was stopped by the addition of 100 μL of0.2 M sodium carbonate solution. One unit of enzyme activity was definedas the amount of enzyme releasing 1 μmol of 4-MU of the substrate perminute under assay conditions. Net values of each reaction werecalculated by subtracting the fluorescence obtained in substrate andenzyme blanks a parallel reaction. A standard curve for free4-methylumbelliferone was used to determine the amount of the productsformed. Enzyme activity was expressed as nmol of 4-methylumbelliferonereleased/min/mg of protein.

Highest activity was obtained with 4-MU-GlcNAc₂ (exochitinase,chitobiosidase activity) followed by 4-MU-GlcNAc₁ (Glucosaminidaseactivity) and lower activities with 4-MU-GlcNAc₃ (endohtinase activity)(Table 6).

TABLE 6 Specific activity for culture supernatant and cell fraction ofthe recombinant, modified chitinase BRLA_Chi 90 using fluorogenic4-methylumbelliferyl substrates Culture Cytoplasmic fraction Substratesupernatant of cell lysate 4-MU-GlcNAc1  3623 ± 38* 1455 ± 564-MU-GlcNAc2 6954 ± 42 2946 ± 39 4-MU-GlcNAc3 1378 ± 28  560 ± 31*Values are mean of the three replicates

Based on the cleavage pattern, chitinases have been classified into twobroad categories-endochitinases and exochitinases. Endochitinases cleavechitin randomly at internal sites to form oligomers of different length,whereas exochitinases are classified as a) chitobiosidases, whichcatalyze the progressive release of chitobiose from the nonreducing endof the chitin chain, and b) β1-4-N-acetylglucosaminidases, which cleavethe oligomeric products of endochitinases and chitobiosidases, typicallychitobioses generating N-acetylglucosamine. Fluorogenic assays showedthat BRLA_Chi 90 exhibits a major exochitinase activity, predominantly achitobiosidase activity and also N-acetylglucosaminidase activity, withsome endochitnase activity. Zymogram analyses and real time ESI-MSstudies also confirmed that BRLA_Chi 90 is a unique, chitinolytic enzymewith a combined exo- and endo-chitinase activity. The combined exo- andendo-chitinase activity results in a synergistic increase in thechitinolytic activity resulting in greater efficacy of chitinase forinsect control and chitin hydrolysis.

The optimal pH for recombinant BRLA_Chi 90 was determined by assayingthe purified enzyme at different pH (3.0-11.0) using appropriate buffersand measuring the relative activity under standard assay conditionsusing fluorogenic 4-methylumbelliferyl substrates. The buffer systemsused were citrate buffer (50 mM, pH 3.0-4.0), acetate (50 mM, pH4.0-5.0), phosphate (50 mM, pH 6.0-8.0), Tris HCl (50 mM 8.0-9.0) andcarbonate-bicarbonate (50 mM, pH 9.0-11.0). The optimum temperature forthe purified recombinant chitinase was determined by performing thestandard assay at temperatures of 25-90° C. in 50 mM sodium acetatebuffer (pH 5.0) and the enzyme activity was measured as relativeactivity.

The optimal temperature of the recombinant chitinase was found to be 55°C. for the N-acetylglucosaminidase and chitotriosidase activity and 60°C. for the chitobiosidase activity (FIG. 7). The recombinant chitinasewas active in a broad range of varied pH (3.0-11.0) with a pH optimum of9.0 (Tris HCl buffer), 10.0 (sodium carbonate-bicarbonate buffer) and4.0 (citrate buffer) for the chitobiosidase, N-acetylglucosaminidase andchitotriosidase activity, respectively (FIG. 8).

Example 9 Evaluation of Secondary Structure and Thermal Stability ofRecombinant, Modified Chitinase, BRLA_Chi 90 by Circular DichroismSpectroscopy (CD)

CD spectrum of recombinant BRLA_Chi 90 showing secondary structure (FIG.9)

CD spectrum of recombinant BRLA_Chi 90 showing thermal unfolding (FIG.10)

CD measurements were conducted using a JASCO-715 spectropolarimeter witha Peltier-type cell holder, which allows for temperature control.Wavelength scans in the far (190 to 260 nm) and the near (260 to 360 nm)UV regions were performed in Quartz SUPRASIL (HELLMA) precision cells of0.1 cm path length. Each spectrum was obtained by averaging five toeight successive accumulations with a wavelength step of 0.2 nm at arate of 20 nm, response time 1 s, and band width 1 nm. Buffer spectrawere accumulated and subtracted from the sample scans. The absorptionspectra were recorded selecting the UV (single) mode of the instrument.CD experiments involving thermal scanning have been carried out in therange from 20 to 90° C., at 215, 220, and 222 nm, and heating scan ratesranging from 0.3 to 2.5 K/min.

Secondary structure of the protein showed 20.47% alpha helix, 27.09%beta sheets, 11.57% turns and 40.93% coils, indicating that BRLA_Chi 90is an alpha-beta protein (FIG. 9). The CD thermal scan studies revealedthat the thermal melting temperature of the recombinant chitinase is66.7° C., indicating that it's a thermostable enzyme (FIG. 10).

Example 10 Time Course Chitin Hydrolysis by Real Time ESI-MS UsingRecombinant, Modified Chitinase BRLA_Chi 90 From Brevibacilluslaterosporus Lak 1210

ESI-MS profile of chitooligosaccharide standards Mass spectroscopicanalysis- using electrospray ionization mass spectrometry (ESI-MS) ofchitoligosaccharides standards with showing their masses—GlcNAc (244.11)GlcNAc₂ (447.23) GlcNAc3 (650.30) GlcNAc4 (853.35) GlcNAc5 (1056.38)GlcNAc6 (1259.4) (FIG. 11)

Real Time ESI-MS of chitin hydrolysis (20 min) using recombinant,modified BRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 12)

Real Time ESI-MS of chitin hydrolysis (1 h) using recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 13)

Real Time ESI-MS of chitin hydrolysis (3 h) using recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 14)

Real Time ESI-MS of chitin hydrolysis (6 h) using recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 15)

Real Time ESI-MS of chitin hydrolysis (12 h) using recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 16)

Real Time ESI-MS of chitin hydrolysis using (24 h) recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 17)

Real Time ESI-MS of chitin hydrolysis (48 h) using recombinant, modifiedBRLA_Chi 90 from Brevibacillus laterosporus Lak 1210 (FIG. 18)

We report on the sensitive monitoring of an enzymatic reaction via timeresolved ESI-MS. Enzymatic reactions are coupled online with MS in whicha continuous-flow system coupled via ion-spray to a mass spectrometerwas used for the detection of the end products by their molecular mass(to charge ratio). Characterization of the recombinant chitinase enzyme,BRLA_Chi 90 by real-time ESI-MS allows an easy and rapid method ofdetermining its chitinase activity using native unlabeled substrateslike chitin flakes, colloidal chitin and in particular, the powderedchitin extracted from crab shells and shrimp shells.

After mixing the substrate (1% colloidal chitin/1% glycol chitin) and 10μl of the purified enzyme (1 mg/ml), the reaction mixture was infusedwith a flow rate of 5 μL/min into the mass spectrometric interface via atubing-connected ( 1/16″×ID 0.13 mm, length 200 mm) syringe(Hamilton-Bonaduz, Switzerland, 100 μL) located in a syringe pump (HugoSachs Elektronik, Hugstetten, Germany). The syringe has to be refilledevery 20 min. Several measurements were carried out at 20±2° C.Experiments were performed using a mass spectrometer from Agilent (SantaClara, Calif., USA), an LC/MSD TOF model equipped with an ESI source.The measurements were carried out in positive ionization mode with 300°C. drying gas temperature, 480 L/min drying gas flow and 15 psignebulizer gas pressure, 4000 V capillary voltage, 60 V skimmer voltageand 215 V fragment or voltage. The mass range was set to 200-2000 m/zand data acquisition was performed at 0.88 cycles/s. The parameters usedfor mass spectrometric detection were optimized with HEWL. The nitrogendrying gas was supplied by a nitrogen generator (nitrogenpurity≥99.5%,). Agilent Technologies (Waldbronn, Germany) software wasused for system control and data acquisition (Analyst QS, LC-MS TOFSoftware, Ver. A.01.00. Extracted ion chromatogram signals were summedfor a time course of 10 min and the time courses were smoothed with aGaussian filter with a width of 400% and a limit of 10.

The end products of time resolved chitin hydrolysis by ESI-MS indicateddiacetylchitobiose as the primary product starting from 20 min to 48 halong with a significant amount of N-acetylglucosamine formed during24-48 h but very low amounts of oligosaccharides (trimers) suggestingthat the enzyme also exhibits endochitinase activity. Since chitobiose(441. 25) was detected in higher amounts as a sodium adduct, it wasdeduced that BRLA_Chi 90 is prevalently an exochitinase withexo-N,N′-diacetylchitobiohydrolase (chitobiosidase) activity. Thedetection of monomer N-acetylglucosamine (226.8) as an end product after24 and 48 h hydrolysis confirmed that the enzyme also possessesN-acetylglucosaminidase activity as confirmed by the zymography assaysand enzyme activity assays using flurogenic 4-Methyl umbelleferylsubstrates. Endochitinase activity of BRLA_Chi 90 was also confirmed byin gel-activity assays, hydrolysis of fluorogenic trisaccharide analogueto triacetylchitotriose and also by the accumulation of trisaccharide byhydrolysis of native colloidal chitin suggesting that it is also anendochitinase. Alternatively, the recombianant enzyme, BRLA_Chi 90 canexhibit exo-N, N′,N″-triacetylchitotriohydrolase activity cleaving thechitotriose to form the major end products chitobiose andN-acetylglucosamine.

Till date, there are not many reports about either chitobioses orchitinases with a combined activity and enzymatic production ofchitobiose and N-acetylglucosamine as major end products, in particularfrom entomopathogens. There are no reports of combined activity ofendochitinase, exoN, N′-diacetylchitobiohydrolase and exoβ-1,4N-acetyl-acetylglucosaminidase activity and/or exo-N,N′,N″-triacetylchitotriohydrolase activity for a single chitinase enzymefrom Brevibacillus laterosporus. To our knowledge, the present study isthe first report of a chitinase with and multiple mechanism forchitinolytic activity from Brevibacillus laterosporus. The presentinvention is also the first report on selective bio production ofindustrially important chitin derivatives, chitobiose and N-acetylglucosamine (NAG) from chitin at the same reaction temperature (55-60°C.), using culture supernatant containing the extracellular recombinantchitinase. Thus, the present invention also pertains to a valuablecost-effective process of N-acetylglucosamine and chitobiose from marinewastes from fishery industries like crustacean shells (crab shells andshrimp shells) using culture supernatant containing the recombinantchitinase enzyme.

Example 11 Evaluation of the Efficacy and Effectiveness of Recombinant,Modified Chitinase BRLA_Chi 90 For Controlling Spodoptera litura TopicalApplication Bioassays—a Scanning Electron Microscopy (SEM) Study of theInsect Cuticle

SEM study of hydrolytic effects of chitinase on the cuticle ofSpodoptera litura (topical bioassays) (FIGS. 19A-19C)

Topical bioassays were performed to observe structural changes on fifthinstar larvae cuticular surfaces were observed by scanning electronmicroscopy after treatment with the recombinant chitinase. Ridge-likestructures could be observed on the intact cuticular surface (treatedwith 20 mM potassium phosphate buffer, pH 7.0) of control larvae. Grossmorphological changes were observed in when cuticular surface wastreated with recombinant chitinase and degradation of the ridge-likestructures was observed, which is suggestive of the hydrolytic effect ofchitinase.

Example 12 Evaluation of the Efficacy and Effectiveness of RecombinantChitinase BRLA_Chi 90 For Controlling Spodoptera litura by DropletFeeding Insect Bioassays—a Histopathological Study of Larval Midgut

Histopathology study of ultrastructural changes in the larval midgutfollowing droplet feeding insect bioassays (FIGS. 20A and 20B)

Droplet feeding oral bioassays were performed on third instar larvae ofSpodoptera litura. Thirty 3^(rd) instar S. litura were injected with 10μl of PBS (different concentrations of purified recombinant chitinase—10ng, 50 ng, 0.1 ng, and 5 μg) into the hemolymph. As a negative control,thirty S. frugiperda larvae were also injected with PBS and theexperiment was repeated three times. The inoculated larvae were placedindividually in petri dishes with castor leaf discs and observed twicedaily until death.

Following ingestion of the chitinase by the larvae of Spodoptera litura,the larvae were agar embedded for cryostat sectioning as per thestandard protocol. The histopathological effects of chitinase on thelarval midgut demonstrated the ultrastructural changes in the midgutwhich include progressive loss of peritrophic membrane, sloughing ofvesicular structures into the lumen and eventual lysis of midgutepithelium of the larvae leading to the death of the larvae.

Example 13 Evaluation of Antifungal Activity Against Fusarium. oxysporumUsing Cell Free Culture Supernatant Containing Recombinant, ModifiedBRLA_Chi 90

Antifungal activity (hyphal extension inhibition) assay showingantagonistic activity of recombinant, modified BRLA_Chi 90 againstFusarium oxysporum (FIG. 21)

Antifungal plate assays were performed by a dual culture method (hyphalextension inhibition assay). The purified chitinase (2 μg/ml in 50 mMphosphate buffer, pH 6.0) was seeded in the center of a well boredPotato Dextrose agar plate and a mycelial plug of the actively growingtest fungus (2 mm) was plugged on either side. The plates were incubatedat 28±3° C. for 4-5 days and examined for the zones of inhibition.Antifungal activity was calculated by measuring the zone of inhibition.The percentage inhibition as calculated as Percentage Inhibition(%)=Diameter of the fungal colony on the control plate−Diameter of thefungal colony on the plate seeded with recombinant chitinase BRLAchi90/Diameter of the fungal colony on the control×100%.

The inhibition growth zone of 36.33±1.69 mm was observed in the testplate seeded with purified chitinase. No antifungal activity wasobserved on the control plate.

In most cases, the antifungal activity is limited to the endochitinases.Most of the antifungal chitinases that have been reported so far areendochitinases. There are very few reports on antifungal activity ofexochitinases, in particular bacterial exochitinases. Inaccessibility ofexochitinases to hydrolyze fungal hyphal cell walls is not clearlyevident. The strong antagonistic activity of recombinant chitinase,BRLA_Chi90 against fungal hyphae could be due to the synergistic actionof exo- and endochitinase activity.

Example 14 Evaluation of Fungal Morphology of Fusarium oxysporum TreatedWith Recombinant, Modified Chitinase BRLA Chi90 by Scanning ElectronMicroscopy (SEM)

SEM study of hyphal morphology of Fusarium oxysporum treated withrecombinant chitinase, BRLA_Chi 90 (FIGS. 22A and 22B)

Herein, Fusarium oxysporum was taken as a model test fungus to study theeffect of recombinant chitinase, BRLA_Chi 90 on hyphal morphology byScanning Electron Microscopy (SEM). Scanning electron microscopy (SEM)studies were carried out to reveal the morphological changes in thehyphae of phytopathogenic fungus, Fusarium oxysporum. The SEM studiesrevealed gross morphological changes in the surface structures of thetreated hyphae compared to the control hyphae, indicating thedegradation of the fungal cell walls.

Example 15 Evaluation of Mode Action of Chitinase and Localization ofFITC-Labeled Recombinant, Modified BRLA_Chi 90 in the Hyphae of Fusariumoxysporum by Fluorescence Microscopy (FIGS. 23A-23D)

Fluorescence microscopy study to evaluate localization of FITC-BRLA_Chi90 in the hyphae of Fusarium oxysporum (FIGS. 24A and 24B)

The mode of action of recombinant chitinase was examined by fluorescentmicroscopy study, using FITC-labelled BRLA_Chi 90. Microscopic analysiswas performed using Zeiss (Oberkochen, Germany) Axio Imager was equippedwith a CCD camera and Plan Neofluar 40× (numeric aperture [NA], 1.3) and63× (NA, 1.25) objective lenses. The excitation of fluorescently labeledproteins was carried out using an HXP metal halide lamp (LEj, Jena,Germany) in combination with a filter set for green fluorescent protein(GFP) (ET470/40BP, ET495LP, and ET525/50BP). Nascent chitin present atthe apical regions of hyphae and in the inner parts of the lateral wallswas more susceptible to chitinase than in the subapical parts of younghyphae but the chitinase was also found to be accumulated in the innercortex of the mature hyphae resulting in complete destruction of thehypha, subsequently. We have observed swelling of the hyphal tips whichsuggested that the growth inhibition is of the consequence of a thinningof the cell wall in the hyphal tip, leading to an imbalance of turgorpressure and wall tension which causes the tip to swell and to burst.

Example 16 Toxicological Studies of Recombinant, Modified ChitinaseBRLA_Chi 90 From Brevibacillus laterosporus Lak 1210 Against Non-TargetAnimals

Bright field photomicrographs of histology of heart in rats (40×, H & Estain) (FIGS. 25A and 25B)

Bright field photomicrographs of histology of kidney in rats (40×, H & Estain) (FIGS. 26A and 26B)

Bright field photomicrographs of histology of liver in rats (40×, H & Estain) (FIGS. 27A and 27B)

Bright field photomicrographs of histology of lungs in rats (40×, H & Estain) (FIGS. 28A and 28B)

Bright field photomicrographs of histology of spleen in rats (40×, H & Estain) (FIGS. 29A and 29B)

TABLE 7 Evaluation of dermal toxicity and dermal irritation in rabbits(Draize's method)-control group Erythema score Edema score Time RabbitsAverage Combined Rabbits Average Combined period 1 2 3 score index 1 2 3score index  1 h 0 0 0 0 0 0 0 0 0 0 24 h 0 0 0 0 0 0 0 0 0 0 48 h 0 0 00 0 0 0 0 0 0 72 h 0 0 0 0 0 0 0 0 0 0  7 d 0 0 0 0 0 0 0 0 0 0 14 d 0 00 0 0 0 0 0 0 0 Primary irritation index: 0.0-1 non-irritant; 1.1-2slightly irritant; 2.1-5 moderately irritant; 5.1-6 severe moderatedirritant; 6.1-8 severe irritant.

TABLE 8 Evaluation of dermal toxicity and dermal irritation in rabbits(Draize's method)-Experimental group tested with the sample, culturesupernatant containing chitinase Erythema score Edema score Time RabbitsAverage Combined Rabbits Average Combined period 1 2 3 score index 1 2 3score index  1 h 0 0 0 0 0 0 0 0 0 0 24 h 0 0 0 0 0 0 0 0 0 0 48 h 0 0 00 0 0 0 0 0 0 72 h 0 0 0 0 0 0 0 0 0 0  7 d 0 0 0 0 0 0 0 0 0 0 14 d 0 00 0 0 0 0 0 0 0 Primary irritation index: 0.0-1 non-irritant; 1.1-2slightly irritant; 2.1-5 moderately irritant; 5.1-6 severe moderatedirritant; 6.1-8 severe irritant.

A new strain, Brevibacillus laterosporus LAK 1210 has been proposed foruse in insect control and it has already been demonstrated that it iseffective biocontrol agent. It is absolutely necessary that thebiocontrol strain of Brevibacillus laterosporus and its chitinase enzymeformulations which is purported to be used in agricultural fields,horticulture farms, forests, forest nurseries is evaluated for interimrisk assessment towards non-target animals. The toxicological studieswere performed to prove the safety in application of formulations ofchitinase enzyme and/or cell suspensions of Brevibacillus laterosporusLAK 1210 as a contact and stomach biopesticide.

Acute oral toxicity and pathogenicity of chitinase formulations ofBrevibacillus laterosporus LAK 1210 and to generate preliminarytoxicology data for the development of a next generation, potentialbiopesticide as a possible alternative to Bt and paves a way for itscommercialization. Low or non-existing toxicity of the enzymesupernatant and cell suspensions of Brevibacillus laterosporus LAK 1210through three different toxicity studies in animal models, such as acuteoral toxicity in a murine model (adult Wistar rats) and a dermalirritation test in rabbits.

On the basis of these results, it was concluded that the chitinaseformulations can contribute to the development of a “low risk”biopesticide.

A. Acute Oral Toxicity Test Using Murine Models (Wistar Rats)

The acute oral toxicity test was conducted according to United StatesEnvironmental Protection Agency (USEPA) guidelines. This study wasconducted on twelve female albino Wistar rats, 7-8-week-old with a bodyweight between 150 and 200 g, obtained from the Central Animal Facility,CCMB, INDIA. They were kept in individual polypropylene cages providedwith clean bedding of rice husk. They were divided into four groups(three experimental groups and one control group). They wereacclimatized for five days prior to dosing under standard housingconditions (temperature: 25±2° C., relative humidity: between 30 and 70%with optimal air changes per hour and 12 h each of dark and light cycle)and provided with standard pelleted feed and U.V. treated water adlibitum.

The animals were acclimatized to the laboratory conditions 5 days priorto the test. The control group was administered sterile PBS. Each of theanimal in the three experimental groups was administered twoconcentrations (5, 10 and 20 mg/kg) of lyophilized extracts of theculture supernatant containing chitinase enzyme. Oral administration wasperformed by gavage for all the animals. The body weight of each animalwas registered on day one of acclimatization, before dosing beginning onthe first day of the study and then twice a week for a period of 14days. During this period, food and water consumption were alsoregistered. At the end of the study, the average gain in weight in gramsper day as well as the average of solids and liquids consumed per daywas obtained in grams or milliliters. Body weight of individual animalswere recorded on the day of dosing, weekly thereafter, twice a week andat termination on day 14.

The treated animals were observed for mortality (twice daily) andclinical signs were recorded to note the onset, duration and reversal(if any) of toxic effects at 1, 2, 4, 8 and 12 h after theadministration of the test substance and once daily thereafter for 14days. The routine cage side observations included changes in skin andfur, eyes respiration, occurrence of secretions and bizarre behavior(e.g. self-mutilation, walking backwards). The behavioral profilestudied included alertness, visual placing, irritability, spontaneousactivity, reactivity and touch response, whereas neurologicalobservations such as straub response, tremors, convulsions, staggeringgait, limb tone, grip strength, corneal reflex and pinna reflex weretaken into consideration. The criteria for autonomic profile includedfindings on pupil size, palpebral opening, exophthalmos, salivation,piloerection. Miscellaneous signs like arching of the back, alopecia,wound, nasal discharge, lacrimation and loose stool were also recordedduring the observations. The clinical signs were graded by a scoringsystem wherein scores for normal, abnormal, subnormal and supernormalresponses were assigned as 4, 0, <4 and >4, respectively and the maximumscore for any response was assigned as 8.

At the end of the study, one animal per group was selected and amacroscopic necropsy was performed, and the gross pathological changes,if any, were recorded, including examination of the outer surface of thebody, all holes and cranial, thoracic and abdominal cavities. Themorphological characteristics of the heart, kidney, spleen and liverwere also assessed. Histopathological examination of liver, kidney,heart, spleen and lungs was considered. The organs were removed, fixedin 10% neutral buffered formalin and processed for paraffin embedding.Sections of 4-6 μm thickness were cut and stained with hematoxylin andeosin (H and E) and observed under light microscope forhistopathological changes.

The treated animals survived throughout the study period and did notreveal any treatment related major abnormal clinical signs or anysignificant pathological changes at the tested dose levels. On necropsy,no abnormalities in the organs were observed in any of the treatedanimals. There were no statistical differences in body weight gainbetween controls and tested groups during the 14-day observation period.The behavioral, neurological and autonomic parameters recorded in termsof graded scores in the treated animals, immediately after theadministration of the chitinase formulations/cell suspensions and dailyonce during the observation period of 14 days, were well within thenormal levels. The gross necroscopy showed no significant changes in theorgans like heart, liver, kidney, spleen and lungs with respect tocontrol (FIGS. 25A, 25B, 26A, 26B, 27A, 28A, 28B, 29A, and 29B).

A. Evaluation of Irritation Tests on Rabbits by Draize's Method

The Draize's skin test was used to evaluate dermal irritation inrabbits. The extracts and cell cultures were administered on the firstday of the study. For each concentration, two rabbits were used applyinga single solution directly on the back with the help of a sterile cottonswab. After application, a patch of surgical gauze with 4 layers and anelastic bandage were fixed thereon in order to prevent the animal fromaccessing the site where the test substances were applied. The totalobservation period lasted 72 h and special attention was given to signsof edema and erythema at 4, 24, 48 and 72 h after removing the patches(using the value scale for skin lesions as described by Draize's).

The erythema values were averaged and added to the edema values (eqn 1)to calculate the Dermal irritation index (DII).

Primary dermal irritation index=x ⁻of erythema+x ⁻of edema   (Eqn 1)

Based on this index for primary dermal irritation, values between 0 and5 are considered to be within the acceptance criteria for the safe useof these extracts and cells on humans. When values range from 6 to 8,the product cannot be utilized on human skin due to it being consideredas an irritant.

In the present example, 0.5 ml of the test sample (culture supernatantcontaining chitinase at a concentration of 1 mg/m1) (Test) and sterile1× PBS (control) was applied to the skin of albino rabbits. After 4hours i.e. the exposure period, the degree of irritation was read andscored at 1 h, 24 h, 48 h, 72 h, 7 and 14 days after the patch removal.

The primary skin irritation index was calculated and came as 0.00 afterpatch removal. Hence, it was concluded that the both the control sample(PBS) and the test sample (culture supernatant containing chitinase) was‘non-irritant’ to the skin of rabbits.

Control sample (0.5 ml sterile PBS) was evenly applied to a small area(approximately 6 cm square) of the closely clipped skin of each of threerabbits. The site of application was covered with a cotton another threerabbits were similarly treated with 0.5 ml of the test sample of culturesupernatant containing chitinase (1 mg/ml). At the termination of 4 hexposure period, the bandages/gauze was removed and treatment sites werecleaned with wet gauze to remove any residual test substance.

Skin reaction at the site of application was subjectively assessed andscored once daily at 1 h, 24 h, 48 h, 72 h; 7 and 14 days after patchremoval (post-test observation period) according to Draize's method.

The reaction at the site of application was assessed and scoredaccording to the following numerical system (Tables 7 and 8).

Example 17 Demonstration of Insecticidal Activity of Recombinant,Modified Chitinase BRLA_Chi 90 From Brevibacillus laterosporus Lak 1210Against Spodoptera litura by Topical Application (Contact) Bioassays

Insecticidal activity of recombinant, modified chitinase, BRLA_Chi 90toward the insect pest, Spodoptera litura was demonstrated by topicalapplication (contact bioassays). Thirty, 3^(rd) instar larvae weretopically treated with culture supernatant recombinant, modifiedchitinase at concentration (5, 10, 15, 20 and 25 μg in 50 mM phosphatebuffer) with 50 mM phosphate buffer (pH 6.0) as a control, allexperiments performed in 5 replicates. The dead larvae were scored at 1,12, 24, and 48 h, after treatment. The results summarized in Table 9,clearly indicate that the recombinant, modified chitinase, BRLA_Chi 90had an effective, insecticidal action on contact. When appliedtopically, the chitinase disrupts the cuticle due to the efficienthydrolyzing activity of the recombinant, modified chitinase, BRLA_Chi90, which could be attributed to the dual enzyme activity and combinedsynergistic effect of the exo- and endochitinase activity of the enzyme.The mortality percentages obtained were 10.9 to 100%, depending upon theconcentration (μg/ml)-contact time (h).

TABLE 9 Mortality (%) of Spodoptera litura against concentration ofrecombinant, modified chitinase, BRLA_Chi 90 (μg/ml) − contact time (h)Concentration (μg/ml) 1 h 12 h 24 h 48 h  0 (control) 0 2.6 ± 0.6  4.1 ±0.12  5.3 ± 0.31  5  9.6 ± 0.39 31.1 ± 0.65 38.2 ± 0.88 50.72 ± 1.12  10 51.7 ± 1.21 62.4 ± 1.45 79.6 ± 1.53  98 ± 2.20 15 69.2 ± 1.42 84.4 ±1.78 95.4 ± 2.02 100 ± 2.50 20 84.5 ± 1.78  100 ± 2.50  100 ± 2.50 100 ±2.50 25  100 ± 2.50  100 ± 2.50  100 ± 2.50 100 ± 2.50

Example 18 Demonstration of Insecticidal Activity of Recombinant,Modified Chitinase BRLA_Chi 90 From Brevibacillus laterosporus Lak 1210Against Third Instar Larvae of Spodoptera litura by Diet IncorporationBioassays

Insecticidal activity of recombinant, modified chitinase, BRLA_Chi 90toward the insect pest, Spodoptera litura was demonstrated by dietincorporation (ingestion) bioassays. The baseline bioassay was performedusing thirty, 3^(rd) instar larvae of Spodoptera litura reared in thelaboratory. The larvae were fed on chitinase-integrated chickpea basedsemisynthetic diet infested with culture supernatant containingrecombinant, modified chitinase added to the diet, at concentration(1.5, 6.0, 24, 48 and 96 μg/g diet) with 50 mM phosphate buffer (pH 6.0)as a control. The larvae were placed in 24 well, tissue culture plate,each larva in a separate well and the plates were incubated at 25° C.with 80% relative humidity. The mortality and the larval weight wererecorded and the dead larvae were scored at 1, 24, 48, after treatment.Probit analysis was done to determine the LC₅₀ values, taking intoaccount, the natural mortality and the statistical analyses of the datawas done using R package, a web based tool. The results indicate thatthe recombinant, modified chitinase, BRLA_Chi 90 had an effective,insecticidal action upon ingestion. The recombinant, modified chitinase,BRLA_Chi 90 enters the gut of the larvae and causes damage to theperitrophic membrane that lines the midgut, thus preventing the larvaefrom feeding, consequently resulting in its death. The alkaline activityof the recombinant, modified enzyme, BRLA_Chi 90 with a pH optimum of9.0, promotes faster lysis of midgut epithelium following the formationof pores and destruction of the peritrophic membrane. The alkalineactivity of the recombinant, modified chitinase also helps in efficientsynergistic action to potentiate the effect of cry toxins, when it isused along with the Bt-biopesticides.

TABLE 10 LC ₅₀ and LC ₉₀ values in μg/g diet (95 ± CL) for differenttime periods for third instar larvae of Spodoptera litura when fed withculture supernatant comprising recombinant, modified chitinase, BRLA_Chi90 Time LC₅₀ Time LC₉₀ Treatment (h) (95% limits) (h) (95% limits)Culture 24 36.6 (25.7-43.2) 24 — supernatant 48 18.9(12.9-22.5) 4861.8(48.1-72.8) containing recombinant, modified chitinase, BRLA_Chi 90—Lethal concentrations were not estimated as it would have requirednotable extrapolation of the recorded data.

What is claimed is:
 1. A recombinant modified chitinase comprising theamino acid sequence of SEQ ID NO:4 or SEQ ID NO:
 5. 2. The recombinantmodified chitinase of claim 1, wherein the recombinant modifiedchitinase exhibits exochitinase activity and endochitinase activity at atemperature from 25° C. to 67° C., and wherein the optimum temperaturefor exochitinase and endochitinase activity is from 55° C. to 60° C. 3.The recombinant modified chitinase of claim 1, wherein the recombinantmodified chitinase exhibits activity in the pH range of 3.0 to 11.0. 4.A composition comprising the recombinant modified chitinase of claim 1.5. The composition of claim 4, further comprising a suitable carrier. 6.A method of treating or modulating phytopathogenic infection in a plantor a part thereof, comprising application of the composition of claim 4to an infected plant or a part thereof.
 7. The method of claim 6,wherein the phytopathogenic infection is a plant disease caused byfungus or insect.
 8. The method of claim 6, wherein the phytopathogenicinfection is a plant disease caused by Fusarium oxysporum.
 9. The methodof claim 6, further comprising application of the compositionconcurrently with Bacillus thuringiensis or Bacillus sphaericus.
 10. Useof the recombinant modified chitinase of claim 1 as biorefining agent,bioremediating agent, chitin valorization agent, biopesticide,biofungicide, bioinsecticide, contact biopesticide or bionematicide. 11.The recombinant modified chitinase of claim 3, wherein the pH is 9.0.12. The method of claim 7, wherein the fungus is selected from Fusarium,Alternaria, Botrytis, Cercospora, Colletotrichum, Erysiphe Monihnia,Pythium, Phytophthora, Puccinia, Rhizotonia, Sclerotium, Trichoderma,Ustilago, Uncinula and Venturia.
 13. The method of claim 7, wherein theinsect is selected from Lepidoptera, Diptera, Coleoptera, Homoptera, andHymenoptera.